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Abstract:

Nucleic acid molecules which encode a branching enzyme from a bacterium
of the genus Neisseria, vectors, host cell, plant cells and plants
containing said nucleic acid molecules as well as starch obtainable from
the plants described are described. Furthermore, an in-vitro method for
producing α-1,6-branched α-1,4-glucans on the basis of
sucrose and a combination of enzymes of an amylosucrase and a branching
enzyme as well as the α-1,6-branched α-1,4-glucans obtainable
by said method are described.

Claims:

1.-3. (canceled)

4. A regulatory region which naturally controls the transcription of a
nucleic acid molecule encoding a branching enzyme in bacterial cells
selected from the group consisting of: (a) nucleic acid molecules
encoding a protein which comprises the amino acid sequence depicted in
SEQ ID NO. 2; (b) nucleic acid molecules comprising the coding region
depicted in SEQ ID NO. 1; (c) nucleic acid molecules encoding a protein
which comprises the amino acid sequence encoded by the insert in plasmid
DSM 12425; (d) nucleic acid molecules comprising the coding region for a
branching enzyme, which is contained in the insert of the plasmid DSM
12425; (e) nucleic acid molecules encoding a protein the sequence of
which has, in the first 100 amino acids, a homology of at least 65% to
the amino acid sequence depicted in SEQ ID NO, 2; (f) nucleic acid
molecules the complementary strand of which hybridizes to a nucleic acid
molecule of (a), (b), (c), (d) and/or (e) and which encode a branching
enzyme from a bacterium of the genus Neisseria; and (g) nucleic acid
molecules the sequence of which deviates from the sequence of a nucleic
acid molecule of (f) due to the degeneracy of the genetic code.

5. The regulatory region according to claim 1, wherein said regulatory
region contains a nucleic acid selected from the group consisting of (a)
nucleotide sequences comprising the nucleotides 1 to 169 of the
nucleotide sequence depicted in SEQ ID NO: 1; (b) the nucleotide sequence
of the regulatory region contained in the insert of the plasmid DSM 12425
or pails thereof; (c) nucleotide sequences hybridizing to the sequences
of (a) or (b) under stringent conditions; and (d) nucleotide sequences
having at least 90% sequence identity to the regulatory regions of (a) or
(b).

6. A regulatory region which naturally controls the transcription of a
nucleic acid molecule encoding a branching enzyme in bacterial cells
selected from the group consisting of: (a) promoter sequences having the
nucleotide sequence of positions 36 to 44, 51 to 55, or 157 to 162 of SEQ
ID NO: 1; and (b) regulatory regions having at least 90% sequence
identity to the promoter sequences of (a).

Description:

[0001] This application is a Divisional of U.S. application Ser. No.
12/905,959 filed on Oct. 15, 2010, which is a Divisional of U.S.
application Ser. No. 11/781,226 filed on Jul. 20, 2007 and now issued as
U.S. Pat. No. 7,833,751, which is a Divisional of U.S. Ser. No.
10/705,195 filed Nov. 10, 2003 and now issued as U.S. Pat. No. 7,732,164,
which is a Divisional of U.S. application Ser. No. 09/807,063, filed on
Jun. 11, 2001 and now issued as U.S. Pat. No. 6,699,694 B1 which is a
national phase under 35 U.S.C. §371 of PCT International Application
No. PCT/EP99/07562 which has an International filing date of Oct. 8,
1999, which designated the United States and on which priority is claimed
under 35 U.S.C. §120, the entire contents of which are hereby
incorporated by reference. This divisional application claims priority
under 35 U.S.C. §119 on Application No. 198 46 635.8 filed in
Germany on Oct. 9, 1998 and Application No. 199 24 342.5 filed in Germany
on May 27, 1999, the entire contents of which are hereby incorporated by
reference.

[0002] The present invention relates to nucleic acid molecules encoding a
branching enzyme from bacteria of the genus Neisseria, vectors, host
cells, plant cells and plants containing such nucleic acid molecules as
well as starch obtainable from the plants described. Furthermore, the
present invention relates to in-vitro methods for the production of
α-1,6-branched α-1,4-glucans on the basis of sucrose and a
combination of enzymes of an amylosucrase and a branching enzyme.
Moreover, the invention relates to glucans that are obtainable by the
method described.

[0003] In many respects, α-1,6-branched α-1,4-glucans are of
enormous interest since they are suitable, for instance, as regards the
production of products in the pharmaceutical and cosmetic industry. They
can be used, e.g. as binding agent for tablets, as carrier substances for
pharmaceutical agents, as packaging material, as carrier substance for
powder additives, as UV-absorbing additive in sun creme and as carrier
substance of flavourings and scents.

[0004] In plants, α-1,6-branched α-1,4-glucans can mainly be
found as amylopectin, a component of starch. In animals and in bacteria,
glucans mainly occur in form of glycogen.

[0005] The polysaccharide starch is formed of chemically uniform basic
building blocks, i.e. the glucose molecules, it is, however, a complex
mixture of different forms of molecules which differ with regard to the
degree of polymerization and branching and which, thus, differ strongly
in their physico-chemical properties. It has to be differentiated between
amylose starch, which is an essentially non-branched polymer of
α-1,4-glycosidically linked glucose units, and the amylopectin
starch, which is a branched polymer in which the branchings are formed
due to the presence of additional α-1,6-glycosidical linkings.
According to textbooks (Voet and Voet, Biochemistry, John Wiley & Sons,
1990), the α-1,6-branchings occur after every 24 to 30 glucose
residues on average, which corresponds to a branching degree of
approximately 3% to 4%. The indications as to the branching degree vary
and depend on the origin of the respective starch (e.g. plant species,
plant variety). In plants that are typically used for the industrial
production of starch the share of amylose in the overall share of starch
varies between 10% and 25%. Various approaches for the production of
α-1,6-branched α-1,4-glucans with different branching degrees
have already been described, with these approaches comprising the use of
(transgenic) plants.

[0006] The heterologous expression of a bacterial glycogen synthase in
potato plants, for instance, leads to a slight decrease of the amylose
content, to an increase in the branching degree and to a modification of
the branching pattern of the amylopectin when compared to wild type
plants (Shewmaker et al., Plant. Physiol. 104 (1994), 1159-1166).
Furthermore, it was observed that the heterologous expression of the
branching enzyme from E. coli (glgB) in amylose-free potato mutants (amf)
(Jacobsen et al., Euphytica 44 (1989), 43-48) leads to amylopectin
molecules which have 25% more branching points (Kortstee et al., Plant J.
10 (1996), 83-90) than the control (amf). For isolating the glucans with
different branching degrees, which were produced in transgenic plants, it
is necessary to carry out additional purification steps in order to
remove, for example, the amylose component. These purification steps are
laborious and, therefore, time-consuming and cost-intensive. Furthermore,
it is not possible to achieve a particular branching degree by means of
these approaches. What is more, due to varying experimental conditions
(environmental factors, location), such in-vivo methods vary considerably
with regard to the quality of the product.

[0007] Glycogen has a higher branching degree than the amylopectin. This
polysaccharide, too, contains α-1,6-branched α-1,4-glucans.
Glycogen also differs from starch in the average length of the
side-chains and in the degree of polymerization. According to textbooks
(Voet and Voet, Biochemistry, John Wiley & Sons, 1990), glycogen
contains, on average, an α-1,6-branching point after every 8 to 12
glucose residues. This corresponds to a branching degree of approximately
8% to 12%. There are varying indications as to the molecular weight of
glycogen, which range from 1 million to more than 1000 millions (D. J.
Manners in: Advances in Carbohydrate Chemistry, Ed. M. L. Wolfrom,
Academic Press, New York (1957), 261-298; Geddes et al., Carbohydr. Res.
261 (1994), 79-89). These indications, too, strongly depend on the
respective organism of origin, its state of nutrition and the kind of
isolation of the glycogen. Glycogen is usually recovered from mussels
(e.g. Mytillus edulis), from mammalian liver or muscles (e.g. rabbit,
rat) (Bell et al., Biochem. J. 28 (1934), 882; Bueding and Orrell, J.
Biol. Chem. 236 (1961), 2854). This renders the production on an
industrial scale very time-consuming and cost-intensive.

[0008] The naturally-occurring α-1,6-branched α-1,4-glucans
described, starch and glycogen, are very different depending on their
content of 1,6-glycosidic branchings. This holds true, amongst others,
with regard to solubility, transparency, enzymatic hydrolysis, rheology,
gel formation and retrogradation properties. For many industrial
applications, such variations in the properties, however, cannot always
be tolerated.

[0009] In-vitro approaches are an alternative to the recovery of
α-1,6-branched α-1,4-glucans from plants or animal organisms.
Compared to in-vivo methods, in-vitro methods are generally better to
control and are reproducible to a greater extent since the reaction
conditions in vitro can be exactly adjusted in comparison with the
conditions in a living organism. This usually allows the production of
invariable products with a high degree of uniformity and purity and,
thus, of high quality, which is very important for any further industrial
application. The preparation of products of a steady quality leads to a
reduction of costs since the procedural parameter that are necessary for
the preparation do not have to be optimised for every preparation set-up.
Another advantage of certain in-vitro methods is the fact that the
products are free of the organisms used in the in-vivo method. This is
absolutely necessary for particular applications in the food and
pharmaceutical industries.

[0010] In general, in-vitro methods can be divided into two different
groups.

[0011] In the first group of methods, various substrates, such as amylose,
amylopectin and glycogen, are subjected to the activity of a branching
enzyme.

[0012] Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625) were able to
prove that using the branching enzyme from potato in connection with the
substrate amylose leads to products that are similar to amylopectin, but
that differ from it in their structure.

[0013] Boyer and Preiss (Biochemistry 16 (1977), 3693-3699) showed, in
addition, that a purified branching enzyme (α-1,4-glucan:
α-1,4-glucan 6-glycosyltransferase) from E. coli may be used to
increase the branching degree of amylose or amylopectin.

[0014] If, however, glycogen from E. coli or rabbit liver is incubated
with the branching enzyme from E. coli, only a slight increase in the
branching degree can be achieved (Boyer and Preiss, loc. cit.).

[0016] Okada et al. made a similar approach (U.S. Pat. No. 4,454,161) to
improve the properties of starch-containing foodstuffs. They incubated
substances, such as amylose, amylopectin, starch or dextrin with a
branching enzyme. This had advantageous effects on the durability of
foodstuffs containing substances that were modified correspondingly.
Furthermore, the patent application EP-A10 690 170 describes the reaction
of jellied starch in an aqueous solution using a branching enzyme. This
results in starches having advantageous properties in the production of
paper.

[0017] However, the aforementioned in-vitro methods have the disadvantage
that they, due to the varying branching degree of the educts (e.g.
starch, amylopectin, etc.), make it impossible to produce uniform
products. In addition, it is not possible to intentionally control the
branching degree and, what is more, the substrates used are quite
expensive.

[0018] The other group of in-vitro methods comprises the de-novo synthesis
of α-1,6-branched α-1,4-glucans starting from various
substrates (glucose-1-phosphate, ADP glucose, UDP glucose) using a
combination of enzymes that consists of a 1,4-glucan-chain-forming enzyme
(phosphorylase, starch synthase, glycogen synthase) and a branching
enzyme.

[0019] Illingwort et al. (Proc. Nat. Acad. Sci. USA 47 (1961), 469-478)
were able to show for an in-vitro method using a phosphorylase A from
muscles (organism unknown) in combination with a branching enzyme
(organism unknown) that the de-novo synthesis of molecules similar to
glycogen using the substrate glucose-1-phosphate was possible. Boyer and
Preiss (loc. cit.) combined the enzymatic activity of a phosphorylase
from rabbit muscles or a glycogen synthase from E. coli with the activity
of a branching enzyme from E. coli using the substrate
glucose-1-phosphate or UDP glucose and in this way generated branched
α-glucans. Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625),
too, analysed the de-novo synthesis of α-1,6-branched
α-1,4-glucans from glucose-1-phosphate using a branching enzyme
from potato in combination with a phosphorylase (1,4-α-D-glucan:
orthophosphate α-glycosyltransferase [EC 2.4.1.1]) from maize. Doi
(Biochimica et Biophysica Acta 184 (1969), 477-485) showed that the
enzyme combination of a starch synthase (ADP-D-glucose:
α-1,4-glucan α-4-glucosyltransferase) from spinach and a
branching enzyme from potato using the substrate ADP glucose resulted in
products similar to amylopectin. Parodi et al. (Arch. Biochem. Biophys.
132 (1969), 11-117) used a glycogen synthase from rat liver combined with
a branching enzyme from rat liver for the de-novo synthesis of branched
glucans from UDP glucose. They obtained a polymer which was similar to
native glycogen and which differs from the polymers that are based on
glucose-1-phosphate.

[0020] This second group of in-vitro methods, too, has the disadvantage
that the substrates, e.g. glucose-1-phosphate, UDP glucose and ADP
glucose, are very expensive. Furthermore, it does not seem to be possible
either to intentionally control the branching degree.

[0021] Buttcher et al. (J. Bacteriol. 179 (1997), 3324-3330) describe an
in-vitro method for the production of water-insoluble α-1,4-glucans
using an amylosucrase and sucrose as substrates. However, only linear
α-1,4-glucans without branchings are synthesized.

[0022] Thus, the technical problem underlying the present invention is to
provide a method allowing the cheap production of α-1,6-branched
α-1,4-glucans for industrial purposes, as well as nucleic acid
molecules encoding the enzymes that may be used in said methods, in
particular branching enzymes.

[0023] This technical problem has been solved by providing the embodiments
characterised in the claims.

[0025] FIG. 2 shows a number of α-1,4-glucans having a varying
degree of α-1,6-branchings which were produced by means of the
method of the invention and which were subsequently dyed with Lugol's
solution.

[0027] FIG. 3 shows a HPLC chromatograph of a highly branched process
product (A) which has been debranched with isoamylase and a rat liver
glycogen sample (B) which has been debranched with isoamylase.

[0028] FIG. 4 shows the scheme of the methylation analysis.

[0029] FIG. 5 shows a diagram of the results of the analysis of sample 7
described in Examples 9 and 10 after one and after two methylation steps.
The values for the 2,3,6-methylation are 96.12% and 96.36%, respectively.

[0031] FIGS. 7 and 8 show gas chromatographs of the samples 3 and 7
described in the Examples.

[0032] FIG. 9 schematically shows the plasmid pBE-fnr-Km.

[0033] FIG. 10 shows an activity gel for the branching enzyme.

[0034] FIG. 11 shows the schematic illustration of an RVA profile.

[0035] FIG. 12 shows the distribution of granule size of the lines 143-13A
and 143-59A compared to the wild type.

[0036] FIG. 13 shows the microscopic magnification of the starch granules
of the lines 143-13A, 143-34A and 143-59A in comparison with the starch
granules of wild type plants (light microscope by Leitz, Germany).

[0037] FIG. 14 shows the gel texture of the starches of different
transgenic lines compared to starches from wild type plants. The texture
was determined by means of a texture analyzer.

[0038] FIG. 15 shows the RVA profile of the starches of the lines 143-11A,
143-13A, 143-59A compared to the wild type.

[0039] FIGS. 16 to 18 show the results of HPLC chromatographies which
represent the pattern of the distribution of the side-chains of the lines
143-WT (=wild type), 143-13A and 143-59A.

[0040] FIG. 19 shows the elution gradient that was used for the
chromatographies depicted in FIGS. 16 to 18.

[0041] FIG. 20 shows the percentage deviation of side-chains having
certain chain lengths of the starches analysed in FIGS. 16 to 18 from the
wild type.

[0042] Therefore, the present invention relates to nucleic acid molecules
encoding a branching enzyme (EC 2.4.1.18) from bacteria of the genus
Neisseria selected from the group consisting of [0043] (a) nucleic acid
molecules encoding a protein which comprises the amino acid sequence
depicted in SEQ ID NO. 2; [0044] (b) nucleic acid molecules comprising
the nucleotide sequence of the coding region which is depicted in SEQ ID
NO. 1; [0045] (c) nucleic acid molecules encoding a protein which
comprises the amino acid sequence that is encoded by the insert of the
plasmid DSM 12425; [0046] (d) nucleic acid molecules comprising the
region of the insert of the plasmid DSM 12425, which encodes a branching
enzyme from Neisseria denitrificans; [0047] (e) nucleic acid molecules
encoding a protein the sequence of which has within the first 100 amino
acids a homology of at least 65% with regard to the sequence depicted in
SEQ ID NO. 2; [0048] (f) nucleic acid molecules the complementary strand
of which hybridizes to a nucleic acid molecule according to (a), (b),
(c), (d) and/or (e) and which encode a branching enzyme from a bacterium
of the genus Neisseria; and [0049] (g) nucleic acid molecules the nucleic
acid sequence of which differs from the sequence of a nucleic acid
molecule according to (f) due to the degeneracy of the genetic code.

[0050] The nucleic acid sequence depicted in SEQ ID NO. 1 is a genomic
sequence which comprises a coding region for a branching enzyme from
Neisseria denitrificans. A plasmid containing said DNA sequence has been
deposited as DSM 12425. By means of said sequence or said molecule, the
person skilled in the art can now isolate homologous sequences from other
Neisseria species or Neisseria strains. He/she may do so using
conventional methods, like screening of cDNA or genomic libraries with
suitable hybridization probes. The homologous sequences may also be
isolated as described in Example 1. Thus, it is possible, for example, to
identify and isolate nucleic acid molecules that hybridize to the
sequence depicted in SEQ ID NO. 1 and that encode a branching enzyme.

[0051] The nucleic acid molecules of the invention may, in principle,
encode a branching enzyme from any bacterium of the genus Neisseria, they
preferably encode a branching enzyme from Neisseria denitrificans.

[0058] Nucleic acid molecules hybridizing to the nucleic acid molecules of
the invention may, in principle, be derived from any bacterium of the
genus Neisseria which expresses a corresponding protein, preferably they
are derived from Neisseria denitrificans. Nucleic acid molecules
hybridizing to the molecules of the invention, may, for instance, be
isolated from genomic or from cDNA libraries. Such nucleic acid molecules
can be identified and isolated using the nucleic acid molecules of the
invention or parts of said molecules or the reverse complements of said
molecules, e.g. by hybridizing according to standard techniques (cf.
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition
(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or
by amplification by means of PCR.

[0059] As hybridization probe nucleic acid molecules can be used which
have exactly or essentially the nucleotide sequence depicted in SEQ ID
NO. 1 or parts thereof. The fragments used as hybridization probes may
also be synthetic fragments which have been produced by means of
conventional synthesis techniques and the sequence of which is
essentially identical to the one of a nucleic acid molecule of the
invention. If genes have been identified and isolated to which the
nucleic acid sequences of the invention hybridize, the sequence should be
determined and the properties of the proteins encoded by said sequence
should be analysed to find out whether they are branching enzymes. For
this purpose, it is particularly suitable to compare the homology on the
nucleic acid and amino acid sequence level and to determine the enzymatic
activity.

[0060] The molecules hybridizing to the nucleic acid molecules of the
invention comprise, in particular, fragments, derivatives and allelic
variants of the above-described nucleic acid molecules encoding a
branching enzyme from bacteria of the genus Neisseria, preferably from
Neisseria denitirificans. In this context, the term "derivative" means
that the sequences of said molecules differ from the sequences of the
aforementioned nucleic acid molecules in one of more positions and have a
high degree of homology to said sequences. Homology, in this context,
means that there is, over the entire length, a sequence identity of at
least 60%, in particular an identity of at least 70%, preferably of more
than 80%, more preferably of more than 90% and most preferably of at
least 95%. The deviations from the above-described nucleic acid molecules
may be caused by, e.g. deletion, addition, substitution, insertion or
recombination.

[0061] Furthermore, homology means that there is a functional and/or
structural equivalence between the respective nucleic acid molecules or
the proteins encoded by these. The nucleic acid molecules which are
homologous to the aforementioned molecules and which are derivatives of
said molecules are usually variations of said molecules which are
modifications that have the same biological functions. These may be both
naturally-occurring variations, e.g. sequences from other Neisseria
species or Neisseria strains and mutations with these mutations occurring
naturally or being introduced by directed mutagenesis. Furthermore, the
variations may be sequences produced synthetically. The allelic variants
may be both naturally-occurring variants and variants that have been
produced synthetically or by recombinant DNA techniques.

[0062] The proteins encoded by the different variants of the nucleic acid
molecules of the invention have certain characteristics in common. These
may include, for instance, biological activity, molecular weight,
immunological reactivity, conformation, etc., as well as physical
properties, such as the migration behaviour in gel electrophoreses,
chromatographic behaviour, sedimentation coefficients, solubility,
spectroscopic properties, stability; pH optimum, temperature optimum,
etc.

[0063] The molecular weight of the branching enzyme from Neisseria
denitrificans is 86.3 kDa, with the molecular weight being deduced from
the amino acid sequence. Hence, the deduced molecular weight of a protein
of the invention preferably ranges from 70 kDa to 100 kDa, more
preferably from 77 kDa to 95 kDa and most preferably it is about 86 kDa.

[0064] The present invention also relates to nucleic acid molecules
encoding a protein having the enzymatic activity of a branching enzyme
with the encoding protein having a homology of at least 65%, preferably
of at least 80% and most preferably of at least 95% in the region of the
N-terminus, preferably in the first 100 amino acids, more preferably in
the first 110 amino acids and most preferably in the first 120 amino
acids to the amino acid sequence depicted in SEQ ID NO. 2.

[0065] In another embodiment, the present application relates to nucleic
acid molecules encoding a protein having activity of a branching enzyme,
the protein comprising at least one, preferably at least 5, more
preferably at least 10 and most preferably at least 20 of the following
peptide motifs:

[0066] The nucleic acid molecules of the invention may be any nucleic acid
molecules, in particular DNA or RNA molecules, e.g. cDNA, genomic DNA,
mRNA, etc. They may be naturally-occurring molecules or molecules
produced by means of genetic or chemical synthesis techniques. They may
be single-stranded molecules which either contain the coding or the
non-coding strand, or they may also be double-stranded molecules.

[0067] Furthermore, the present invention relates to nucleic acid
molecules which are at least 15, preferably more than 50 and most
preferably more than 200 nucleotides in length, these nucleic acid
molecules specifically hybridizing to at least one nucleic acid molecule
of the invention. In this context, the term "specifically hybridizing"
means that said molecules hybridize to nucleic acid molecules encoding a
protein of the invention, however, not to nucleic acid molecules encoding
other proteins. The term "hybridizing" means preferably hybridizing under
stringent conditions (see above). In particular, the invention relates to
nucleic acid molecules which hybridize to transcripts of nucleic acid
molecules of the invention and which, thus, can prevent the translation
thereof. Such nucleic acid molecules which specifically hybridize to the
nucleic acid molecules of the invention may, for instance, be components
of anti-sense constructs or ribozymes or may be used as primers for
amplification by means of PCR.

[0068] Moreover, the invention relates to vectors, in particular plasmids,
cosmids, viruses, bacteriophages and other vectors that are usually used
in genetic engineering and that contain the above-described nucleic acid
molecules of the invention.

[0069] In a preferred embodiment, the nucleic acid molecules contained in
the vectors are linked in sense-orientation to regulatory elements
guaranteeing expression in prokaryotic or eukaryotic cells. In this
context, the term "expression" means both transcription or transcription
and translation.

[0070] The expression of the nucleic acid molecules of the invention in
prokaryotic cells, e.g. in Escherichia coli, allows, for instance, a more
exact characterisation of the enzymatic activities of the proteins
encoded. In addition, it is possible to introduce various mutations into
the nucleic acid molecules of the invention by means of conventional
techniques of molecular biology (cf. e.g. Sambrook et al., loc. cit.).
This leads to the synthesis of proteins the properties of which have
optionally been modified. It is also possible to produce deletion mutants
by continued deletion of the 5' or 3' end of the encoding DNA sequence,
which results in the generation of nucleic acid molecules leading to the
synthesis of correspondingly shortened proteins. Moreover, it is possible
to introduce point mutations at positions that influence, for instance,
the enzyme activity or the regulation of the enzyme. In this way, mutants
may be generated that have a modified KM value or that are no longer
subjected to the usual regulation mechanisms in the cells via allosteric
regulation or covalent modification. In addition, mutants may be produced
which have a modified substrate or product specificity. Furthermore,
mutants may be produced which have a modified activity-temperature
profile. The genetic manipulation in prokaryotic cells may be carried out
according to methods known to the skilled person (cf. Sambrook et al.,
loc. cit.).

[0071] Regulatory sequences for the expression in prokaryotic organisms,
e.g. E. coli, and in eukaryotic organisms have been sufficiently
described in the literature, in particular sequences for the expression
in yeast, such as Saccharomyces cerevisiae. Methods in Enzymology 153
(1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987),
516-544) give an overview of various systems for the expression for
proteins in various host organisms.

[0072] Preferably, the nucleic acid molecule of the invention which has
been inserted in a vector of the invention is modified in such a way that
it is easier to isolate the encoded protein from the culture medium after
it had been expressed in a suitable host organism. There is, for
instance, the possibility of expressing the encoded branching enzyme as a
fusion protein together with a further polypeptide sequence the specific
binding properties of which allow the isolation of the fusion protein by
means of affinity chromatography (cf. Chong et al., Gene 192 (1997),
271-281; Hopp et al., Bio/Technology 6 (1988), 1204-1210; Sassenfeld,
Trends Biotechnol. 8 (1990), 88-93).

[0073] Furthermore, the nucleic acid molecule contained in vector of the
invention is preferred to comprise nucleotide sequences which allow the
secretion of the branching enzyme into the culture medium. Preferably, a
sequence is used which codes for the signal peptide of the α-CGTase
from Klebsiella oxytoca M5A1 (Fiedler et al., J. Mol. Biol. 256 (1996),
279-291; Genebank acc. no. X86014, CDS 11529-11618). The recovery and the
purification is made easier by the secretion of the enzyme into the
culture medium. A disruption of the cells is avoided and the enzyme can
be recovered from the culture medium with conventional methods, such as
dialysis, osmosis, chromatographic methods, etc. being used for removing
residuary components of the culture medium.

[0074] Furthermore, the vectors of the invention may comprise other
functional units which may bring about a stabilisation of the vector in a
host organism, such as a bacterial replication origin or the 2μ-DNA
for the stabilisation in S. cerevisiae.

[0075] In another embodiment, the invention relates to host cells, in
particular to prokaryotic or eukaryotic cells which have been transformed
with a nucleic acid molecule or a vector as described above, as well as
to cells which are derived from said host cells and which contain the
described nucleic acid molecules or vectors. The host cells may be
bacterial cells (e.g. E. coli) or fungal cells (e.g. yeast, in particular
S. cerevisiae), as well as plant or animal cells. The term "transformed"
means that the cells of the invention have been genetically modified with
a nucleic acid molecule of the invention in so far as they contain at
least one nucleic acid molecule of the invention in addition to their
natural genome. Said nucleic acid molecule may be present free in the
cell, optionally as self-replicating molecule, or it may be stably
integrated into the genome of the host cell.

[0076] The host cells are preferred to be microorganisms. Within the
present invention, such microorganisms may be all bacteria and all
protista (e.g. fungi, in particular yeasts and algae) as have been
defined, for instance, in Schlegel "Allgemeine Mikrobiologie" (Georg
Thieme Verlag (1985), 1-2).

[0077] The host cells of the invention are particularly preferred to be
plant cells. In principle, these may include plant cells from any plant
species, i.e. both from monocotyledonous and dicotyledonous plants.
Preferably, said cells are plant cells from agricultural useful plants,
i.e. plants that people cultivate for nutritional or technical purposes,
in particular, for industrial purposes. The invention preferably relates
to plants cells from fibre-forming plants (e.g. flax, hemp, cotton),
oil-storing plants (e.g. rape, sunflower, soy bean), sugar-storing plants
(e.g. sugar beat, sugar cane, sugar millet, banana) and protein-storing
plants (e.g. leguminoses).

[0080] Moreover, the present invention relates to a method for producing a
branching enzyme from bacteria of the genus Neisseria. In said method,
the host cells of the invention are cultivated under conditions allowing
the protein to be expressed and the protein is recovered from the
culture, i.e. from the cells and/or the culture medium. Preferably, a
host organism that secretes the branching enzyme is used.

[0081] Furthermore, the present invention relates to a method for
producing a branching enzyme from bacteria of the genus Neisseria with
the protein being produced in an in-vitro transcription and translation
system using a nucleic acid molecule of the invention. The person skilled
in the art is familiar with such systems.

[0082] The invention also relates to proteins which are encoded by the
nucleic acid molecules of the invention or which are obtainable by a
method of the invention.

[0083] Furthermore, the present invention relates to antibodies which
specifically recognise a protein of the invention. These antibodies may
be, for instance, monoclonal or polyclonal antibodies. They may also be
fragments of antibodies which recognise the proteins of the invention.
The person skilled in the art is familiar with methods for producing said
antibodies or fragments.

[0084] Furthermore, the present invention relates to the use of a
branching enzyme of the invention for the production of
α-1,6-branched α-1,4-glucans in in-vitro systems.

[0085] In particular, the present invention also relates to transgenic
plant cells which contain the nucleic acid molecules or vectors of the
invention. Preferably, the cells of the invention are characterised in
that the nucleic acid molecule of the invention which has been introduced
is stably integrated into the genome and is controlled by a promoter
active in plant cells.

[0086] There is a plurality of promoters or regulatory elements at
disposal for expressing a nucleic acid molecule of the invention in plant
cells. In principle, all promoters, enhancers, terminators, etc. that are
active in plants are regulatory elements for the expression in plant
cells. Basically any promoter which is functional in the plants selected
for the transformation can be used. With regard to the plant species
used, the promoter can be homologous or heterologous. Said promoter may
be selected in such a way that the expression takes place in a
constitutive manner or only in a particular tissue, at a certain time in
the development of the plant or at a time that is determined by external
influence. Examples of suitable promoters are the 35S promoter of the
cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812 or
U.S. Pat. No. 5,352,605), which ensures a constitutive expression in all
tissues of a plant, and the promoter construct described in WO/9401571.
The ubiquitin promoter (cf. e.g. U.S. Pat. No. 5,614,399) and the
promoters of the polyubiquitin genes from maize (Christensen et al., loc.
cit.) are further examples. However, also promoters which are only
activated at a time determined by external influence (cf. e.g.
WO/9307279) can be used. Promoters of heat shock proteins allowing a
simple induction may be of particular interest. Furthermore, promoters
can be used which lead to the expression of downstream sequences in a
certain tissue of the plant, e.g. in photosynthetically active tissue.
Examples thereof are the ST-LS1 promoter (Stockhaus et al., Proc. Natl.
Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),
2445-2451), the Ca/b promoter (cf. e.g. U.S. Pat. No. 5,656,496, U.S.
Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992),
3654-3658) and the Rubisco SSU promoter (cf. e.g. U.S. Pat. No. 5,034,322
and U.S. Pat. No. 4,962,028). In addition, promoters that are active in
the starch-storing organs of plants to be transformed are to be
mentioned. It is, for instance, the maize kernels in maize, whereas in
potatoes, it is the tubers. For over-expressing the nucleic acid
molecules of the invention in potato, the tuber-specific patatin gene
promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can, for
example, be used. Seed-specific promoters have already been described for
various plant species. The USP promoter from Vicia faba, which guarantees
a seed-specific expression in V. faba and other plants (Fiedler et al.,
Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet.
225 (1991), 459-467) is an example thereof.

[0087] Moreover, fruit-specific promoters as described in WO 91/01373 can
also be used. Promoters for an endosperm-specific expression, such as the
glutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng
et al., Plant J. 4 (1993), 357-366), the HMG promoter from wheat, the USP
promoter, the phaseolin promoter or promoters of zein genes from maize
(Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant
Mol. Biol. 15 (1990), 81-93) are particularly preferred. By means of
endosperm-specific promoters it is possible to increase the amounts of
transcripts of the nucleic acid molecules of the invention in the
endosperm in comparison with the endosperm of corresponding wild type
plants.

[0089] In addition, there may be a terminator sequence which is
responsible for the correct termination of the transcription and the
addition of a poly-A tail to the transcript having the function of
stabilising the transcripts. Such elements have been described in the
literature (cf. e.g. Gielen et al., EMBO J. 8 (1989), 23-29) and may be
exchanged at will.

[0090] Therefore, it is possible to express the nucleic acid molecules of
the invention in plant cells.

[0091] Thus, the present invention also relates to a method for producing
transgenic plant cells comprising introducing a nucleic acid molecule or
a vector of the invention into plant cells. The person skilled in the art
has various plant transformation systems at disposal, e.g. the use of
T-DNA for transforming plant cells has been examined extensively and has
been described in EP-A-120 516; Hoekema: The Binary Plant Vector System,
Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley,
Crit. Rev. Plant. Sci., 4, 1-46 and An, EMBO J. 4 (1985), 277-287.

[0092] For transferring the DNA in the plant cells, plant explants may
suitably be co-cultivated with Agrobacterium tumefaciens or Agrobacterium
rhizogenes. Whole plants may then be regenerated from the infected plant
material (e.g. parts of leaves, stem segments, roots and protoplasts or
plant cells cultivated in suspensions) in a suitable medium which can
contain antibiotics or biocides for selecting transformed cells. The
plants obtained in that way can then be examined for the presence of the
DNA introduced. Other possibilities of introducing foreign DNA using the
biolistic method or by protoplast transformation are known (cf.
Willmitzer, L. 1993 Transgenic plants. In: Biotechnology, A Multi-Volume
Comprehensive Treatise (H. J. Rehm, G. Reed, A. Piihler, P. Stadler,
eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

[0093] Alternative systems for transforming monocotyledonous plants are
the transformation by means of the biolistic method, the electrically or
chemically induced DNA absorption in protoplasts, the electroporation of
partially permeabilised cells, the microinjection of DNA in the
inflorescence, the microinjection of DNA in microspores and pro-embryos,
the DNA absorption through germinating pollens and the DNA absorption in
embryos by swelling (cf. e.g. Lusardi, Plant J. 5 (1994), 571-582;
Paszowski, Biotechnology 24 (1992), 387-392).

[0095] In the past, three of the above transformation systems could be
established for various cereals: the electroporation of tissue, the
transformation of protoplasts and the DNA transfer by particle
bombardment in regenerable tissue and cells (for an overview see Jane,
Euphytica 85 (1995), 35-44). The transformation of wheat has been
described several times in the literature (for an overview see
Maheshwari, Critical Reviews in Plant Science 14 (2) (1995), 149-178).

[0098] For expressing the nucleic acid molecules of the invention in
plants it is, in principle, possible for the synthesized protein to be
located in any compartment of the plant cell. The coding region must
optionally be linked to DNA sequences which guarantee the localisation in
the respective compartment in order to achieve localisation in a
particular compartment. Such sequences are known (cf. e.g. Braun, EMBO J.
11 (1992), 3219-3227; Sonnewald, Plant J. 1 (1991), 95-106; Rocha-Sosa,
EMBO J. 8 (1989), 23-29).

[0099] As plastidial signal sequence, for instance, the one of
ferrodoxin:NADP+ oxidoreductase (FNR) from spinach can be used. Said
sequence contains the 5' non-translated region and the flanking transit
peptide sequence of the cDNA of the plastidial protein ferrodoxin:NADP+
oxidoreductase from spinach (nucleotide -171 to +165; Jansen et al.,
Current Genetics 13 (1988), 517-522).

[0100] Furthermore, the transit peptide of the waxy protein from maize
plus the first 34 amino acids of the mature waxy protein (Klosgen et al.,
Mol. Gen. Genet. 217 (1989), 155-161) may also be used as plastidial
signal sequence. In addition, the transit peptide of the waxy protein
from maize (cf. above) may also be used without the 34 amino acids of the
mature waxy protein.

[0102] Therefore, the present invention also relates to transgenic plant
cells that were transformed with one or more of the nucleic acid
molecule(s) of the invention, as well as to transgenic plant cells that
are derived from cells transformed in such a way. Such cells contain one
or more nucleic acid molecule(s) of the invention with said molecule(s)
preferably being linked to regulatory DNA elements which guarantee the
transcription in plant cells, in particular with a promoter. Such cells
can be differentiated from naturally-occurring plant cells in that they
contain at least one nucleic acid molecule of the invention.

[0103] The transgenic plant cells may be regenerated to whole plants using
techniques well-known to the person skilled in the art. The plants
obtainable by means of regeneration of the transgenic plant cells of the
invention are also a subject matter of the present invention.

[0107] In a preferred embodiment, the cells of the plants of the invention
have an increased activity of the protein of the invention in comparison
with corresponding plant cells of wild type plants that have not been
genetically modified. These are preferably cells of starch-storing
tissue, in particular cells of tubers or of the endosperm, most
preferably cells of potato tubers or the endosperm of maize, wheat or
rice plants.

[0108] Within the meaning of the present invention, the term "increase of
the activity" means an increase in the expression of a nucleic acid
molecule of the invention which encodes a protein with branching enzyme
activity, an increase in the amount of protein with branching enzyme
activity and/or an increase in the activity of a protein with branching
enzyme activity in the plants.

[0109] The increase in the expression can, for instance, be determined by
measuring the amount of transcripts coding for said proteins, e.g. by
means of Northern blot analysis or RT-PCR. In this context, the term
"increase" preferably means an increase in the amount of transcripts by
at least 10%, preferably by at least 20%, more preferably by at least 50%
and most preferably by at least 75% in comparison with plant cells that
have not been genetically modified.

[0110] The amount of proteins with branching enzyme activity may, for
example, be determined by Western blot analysis. In this context, the
term "increase" preferably means that the amount of proteins with
branching enzyme activity is increased by at least 10%, preferably by at
least 20%, more preferably by at least 50% and most preferably by at
least 75% in comparison with corresponding cells that have not been
genetically modified.

[0111] An increase in the activity of the branching enzyme can, for
instance, be determined according to the method described in Lloyd et al.
(Biochem. J. 338 (1999), 515-521). In this context, the term "increase"
preferably means that the branching enzyme activity is increased by at
least 10%, preferably by at least 20%, more preferably by at least 50%
and most preferably by at least 75%.

[0112] Surprisingly, it was found that plants containing plant cells of
the invention with an increased activity of a branching enzyme synthesize
a modified starch compared to corresponding wild type plants that have
not been genetically modified. The modified starch may, for instance, be
modified with regard to its physio-chemical properties, in particular the
amylose/amylopectin ratio, the branching degree, the average chain
length, the phosphate content, the viscosity, the size of the starch
granule, the distribution of the side-chains and/or the form of the
starch granule in comparison with starch synthesized in wild type plants.
As a consequence, this modified starch is more suitable for particular
purposes.

[0113] Furthermore, it was surprisingly found that in plant cells in which
the activity of the branching enzyme of the invention is increased, the
composition of the starch is modified in such a way that it has a higher
gel texture and/or a reduced phosphate content and/or a reduced peak
viscosity and/or a reduced pastification temperature and/or a reduced
size of the starch granule and/or a modified distribution of the
side-chains in comparison with starch from corresponding wild type
plants.

[0114] In this context, the term "increased gel texture" means an increase
by at least 10%, preferably by at least 50%, more preferably by at least
100%, by at least 200% and most preferably by at least 300% in comparison
with the gel texture of starch from wild type plants. The gel texture is
determined as described below.

[0115] Within the meaning of the present invention, the term "reduced
phosphate content" means that the overall content of covalently bound
phosphate and/or the content of phosphate in the C-6 position of the
starch synthesized in the plant cells of the invention is reduced by at
least 20%, preferably by at least 40%, more preferably by at least 60%
and most preferably by at least 80% in comparison with the starch from
plant cells of corresponding wild type plants.

[0116] The overall phosphate content or the content of phosphate in the
C-6 position may be determined according to the method as described
below.

[0117] Within the meaning of the present invention, the term "reduced peak
viscosity" means that the peak viscosity is reduced by at least 10%,
preferably by at least 25%, more preferably by at least 50% and most
preferably by at least 75% in comparison with the peak viscosity of
starches from wild type plants.

[0118] Within the meaning of the present invention, the term "reduced
pastification temperature" means that the pastification temperature is
reduced by at least 0.5° C., preferably by at least 1.0°
C., more preferably by at least 2.0° C., most preferably by at
least 3.0° C. in comparison with the pastification temperature of
starches from wild type plants.

[0119] The peak viscosity and the pastification temperature can be
determined with a Rapid Visco Analyzer in the manner described below.

[0120] The skilled person is familiar with the terms "peak viscosity" and
"pastification temperature".

[0121] The term "reduced size of the starch granule" means that the
percentage proportion of the starch granules having a size of up to 15
μm is increased by at least 10%, preferably by at least 30%, more
preferably by at least 50%, 100% and most preferably by at least 150% in
comparison with wild type plants.

[0122] The size of the starch granules is determined by means of a
photosedimentometer of the type "Lumosed" by Retsch, GmbH, Germany in the
manner described below.

[0123] In this context, the term "modified distribution of the
side-chains" means that the proportion of side-chains with a DP of 6 to 9
is increased by at least 25%, preferably by at least 50%, more preferably
by at least 100% and most preferably by at least 200% in comparison with
the proportion of side-chains with a DP of 6 to 9 of amylopectin from
wild type plants.

[0124] In another embodiment of the invention, the term a "modified
distribution of side-chains" means that the proportion of side-chains
with a DP of 6 to 8, preferably of 6 to 7 is increased by at least 25%,
preferably by at least 50%, more preferably by at least 100% and most
preferably by at least 200% in comparison to the proportion of
side-chains with the corresponding degree of polymerization of
amylopectin from wild type plants.

[0125] The proportion of side chains is established by determining the
percentage proportion of a particular side-chain with regard to the
overall share of all side-chains. The overall share of all side-chains is
established by determining the overall area below the peaks which
represent the polymerization degrees of DP 6 to 30 in the HPLC
chromatograph. The percentage proportion of a particular side-chain with
regard to the overall share of all side-chains is established by
determining the ratio of the area below the peak that represents said
side-chain in the HPLC chromatograph to the overall area. Preferably, the
program AI-450, version 3.31 by Dionex, USA, is used.

[0126] In another embodiment, the present invention relates to a starch
the amylopectin of which has side-chains with a DP of 5 compared to the
amylopectin of starches of wild type plants.

[0127] Furthermore, the present invention relates to a method for
producing a transgenic plant which synthesizes a modified starch, wherein
[0128] (a) a plant cell is genetically modified by introducing a nucleic
acid molecule of the invention and/or a vector of the invention the
presence or expression of which leads to an increase in the activity of a
protein having the activity of a branching enzyme; [0129] (b) a plant is
regenerated from the cell produced according to step (a); and [0130] (c)
optionally further plants are produced from the plant produced according
to step (c).

[0131] In a preferred embodiment of the method, the starch is modified in
such a way that it has an increased gel texture and/or a reduced
phosphate content and/or a reduced peak viscosity and/or a reduced
pastification temperature and/or a reduced size of the starch granules
and/or a modified distribution of the side-chains compared to the starch
of corresponding wild type plants.

[0133] As regards the genetic modification introduced according to step
(a), the same applies as has been explained in a different context with
regard to the plants of the invention.

[0134] The regeneration of plants according to step (b) can be achieved by
methods known to the skilled person.

[0135] Further plants according to step (b) of the method of the invention
may, for instance, be produced by vegetative propagation (e.g. by means
of cuttings, tubers or through callus culture and regeneration of whole
plants) or by sexual reproduction. Preferably, the sexual reproduction is
controlled, i.e. selected plants having particular properties are
cross-bred and propagated.

[0136] The present invention also relates to the plants obtainable by the
method of the invention.

[0137] The present invention also relates to propagation material of
plants of the invention as well as of the transgenic plants produced
according to the method of the invention. In this context, the term
"propagation material" comprises those components of the plant that are
suitable for producing progenies in a vegetative or generative way are,
for example, cuttings, callus cultures, rhizomes or tubers are suitable
for the vegetative propagation. Other propagation material comprises, for
example, fruit, seeds, seedlings, protoplasts, cell cultures, etc. The
propagation material is preferred to be tubers and seeds.

[0138] Starch obtainable from the transgenic plant cells and plants of the
invention as well as from the propagation material is a further subject
matter of the invention.

[0139] Due to the expression of a nucleic acid molecule of the invention
or of a vector of the invention, the presence of expression of which
leads to an increase in the activity of a branching enzyme compared to
plant cells of wild type plants that have not been genetically modified,
the transgenic plant cells and plants of the invention synthesize a
starch which is modified with regard to its physio-chemical properties,
in particular with regard to gel texture and/or pastification behaviour
and/or the size of the starch granule and/or the phosphate content and/or
the distribution of the side-chains in comparison with starch synthesized
in wild type plants.

[0140] Moreover, the present invention also relates to starches
characterised in that they have an increased gel texture and/or a reduced
phosphate content and/or a reduced peak viscosity and/or a reduced
pastification temperature and/or a reduced sized of the starch granules
and/or a modified distribution of the side-chains.

[0141] In a particularly preferred embodiment, the present invention
relates to potato starches.

[0143] In addition, the present invention relates to a method for
producing a modified starch comprising the step of extracting the starch
from a plant (cell) of the invention as described above and/or from
starch-storing parts of such a plant. Preferably, such a method also
comprises the step of harvesting the cultivated plants and/or the
starch-storing parts of said plants before the starch is extracted and,
more preferably, also the step of cultivating plants of the invention
prior to harvesting them. The skilled person is familiar with methods for
extracting the starch of plants or of starch-storing parts of plants.
Furthermore, methods for extracting the starch from various
starch-storing plants have been described, e.g. in "Starch: Chemistry and
Technology (ed.: Whistler, BeMiller and Paschall (1994), 2nd
edition, Academic Press Inc. London Ltd.; ISBN 0-12-746270-8; cf. e.g.
chapter XII, page 412-468: Maize and Sorghum Starches: Production; by
Watson; chapter XIII, page 469-479; Tapioca, Arrow Root and Sago
Starches: Production; by Corbishley and Miller; chapter XIV, page
479-490: Potato Starch: Production and Applications; by Mitch; chapter
XV, page 491 to 506: Wheat Starch Production, Modification and
Applications; by Knight and Oson; and chapter XVI, page 507-528: Rice
Starch: Production and Applications; by Rohmer and Klem; Maize Starch:
Eckhoff et al., Cereal Chem. 73 (1996), 54-57, the extraction of maize
starch on an industrial scale is usually achieved by means of the
so-called wet milling)). Appliances that are usually used for methods for
extracting starch from plant material include separators, decanters,
hydrocyclones, spray dryers and fluid-bed dryers.

[0144] Starch obtainable by the method described above is also a subject
matter of the present invention.

[0145] The starches of the invention can be modified according to methods
known to the person skilled in the art and are suitable for various
applications in the foodstuff or non-foodstuff industry in an unmodified
or modified form.

[0146] In principle, possibilities of use can be divided into two large
areas. One area comprises hydrolysis products of the starch, mainly
glucose and glucan building blocks obtained via enzymatic or chemical
methods. They serve as starting material for further chemical
modifications and processes such as fermentation. For a reduction of
costs the simplicity and inexpensive carrying out of a hydrolysis method
can be of importance. At present, the method is essentially enzymatic
with use of amyloglucosidase. It would be possible to save costs by
reducing use of enzymes. This could be achieved by changing the structure
of the starch, e.g. surface enlargement of the granule, easier
digestibility due to low branching degree or a steric structure limiting
the accessibility for the enzymes used.

[0147] The other area where starch is used as so-called native starch due
to its polymeric structure can be subdivided into two further fields of
application:

1. Use in Foodstuffs

[0148] Starch is a classic additive for various foodstuffs, in which it
essentially serves the purpose of binding aqueous additives and/or causes
an increased viscosity or an increased gel formation. Important
characteristic properties are flowing and sorption behaviour, swelling
and pastification temperature, viscosity and thickening performance,
solubility of the starch, transparency and paste structure, heat, shear
and acid resistance, tendency to retrogradation, capability of film
formation, resistance to freezing/thawing, digestibility as well as the
capability of complex formation with e.g. inorganic or organic ions.

2. Use in Non-Foodstuffs

[0149] The other major field of application is the use of starch as an
adjuvant in various production processes or as an additive in technical
products. The major fields of application for the use of starch as an
adjuvant are, first of all, the paper and cardboard industry. In this
field, the starch is mainly used for retention (holding back solids), for
sizing filler and fine particles, as solidifying substance and for
dehydration. In addition, the advantageous properties of starch with
regard to stiffness, hardness, sound, grip, gloss, smoothness, tear
strength as well as the surfaces are utilized.

2.1 Paper and Cardboard Industry

[0150] Within the paper production process, a differentiation can be made
between four fields of application, namely surface, coating, mass and
spraying.

[0151] The requirements on starch with regard to surface treatment are
essentially a high degree of brightness, corresponding viscosity, high
viscosity stability, good film formation as well as low formation of
dust. When used in coating the solid content, a corresponding viscosity,
a high capability to bind as well as a high pigment affinity play an
important role. As an additive to the mass rapid, uniform, loss-free
dispersion, high mechanical stability and complete retention in the paper
pulp are of importance. When using the starch in spraying, corresponding
content of solids, high viscosity as well as high capability to bind are
also significant.

2.2 Adhesive Industry

[0152] A major field of application is, for instance, in the adhesive
industry, where the fields of application are subdivided into four areas:
the use as pure starch glue, the use in starch glues prepared with
special chemicals, the use of starch as an additive to synthetic resins
and polymer dispersions as well as the use of starches as extenders for
synthetic adhesives. 90% of all starch-based adhesives are used in the
production of corrugated board, paper sacks and bags, composite materials
for paper and aluminum, boxes and wetting glue for envelopes, stamps,
etc.

2.3 Textiles and Textile Care Products

[0153] Another possible use as adjuvant and additive is in the production
of textiles and textile care products. Within the textile industry, a
differentiation can be made between the following four fields of
application: the use of starch as a sizing agent, i.e. as an adjuvant for
smoothing and strengthening the burring behaviour for the protection
against tensile forces active in weaving as well as for the increase of
wear resistance during weaving, as an agent for textile improvement
mainly after quality-deteriorating pretreatments, such as bleaching,
dying, etc., as thickener in the production of dye pastes for the
prevention of dye diffusion and as an additive for warping agents for
sewing yarns.

2.4 Building Industry

[0154] Furthermore, starch may be used as an additive in building
materials. One example is the production of gypsum plaster boards, in
which the starch mixed in the thin plaster pastifies with the water,
diffuses at the surface of the gypsum board and thus binds the cardboard
to the board. Other fields of application are admixing it to plaster and
mineral fibers. In ready-mixed concrete, starch may be used for the
deceleration of the sizing process.

2.5 Ground Stabilisation

[0155] Furthermore, the starch is advantageous for the production of means
for ground stabilisation used for the temporary protection of ground
particles against water in artificial earth shifting. According to
state-of-the-art knowledge, combination products consisting of starch and
polymer emulsions can be considered to have the same erosion- and
encrustation-reducing effect as the products used so far; however, they
are considerably less expensive.

2.6 Use in Plant Protectives and Fertilizers

[0156] Another field of application is the use of starch in plant
protectives for the modification of the specific properties of these
preparations. For instance, starch is used for improving the wetting of
plant protectives and fertilizers, for the dosed release of the active
ingredients, for the conversion of liquid, volatile and/or odorous active
ingredients into microcristalline, stable, deformable substances, for
mixing incompatible compositions and for the prolongation of the duration
of the effect due to a reduced disintegration.

2.7 Drugs, Medicine and Cosmetics Industry

[0157] Starch may also be used in the fields of drugs, medicine and in the
cosmetics industry. In the pharmaceutical industry, starch may be used as
a binder for tablets or for the dilution of the binder in capsules.
Furthermore, starch is suitable as disintegrant for tablets since, upon
swallowing, it absorbs fluid and after a short time it swells so much
that the active ingredient is released. For qualitative reasons, medical
lubricating and vulnerary dusting powders are further fields of
application. In the field of cosmetics, the starch may for example be
used as a carrier of powder additives, such as scents and salicylic acid.
A relatively extensive field of application for the starch is toothpaste.

2.8 Starch as an Additive in Coal and Briquettes

[0158] Starch can also be used as an additive in coal and briquettes. By
adding starch, coal can be quantitatively agglomerated and/or briquetted
in high quality, thus preventing premature disintegration of the
briquettes. Barbecue coal contains between 4 and 6% added starch,
calorated coal between 0.1 and 0.5%. Furthermore, starch is suitable as a
binding agent since adding it to coal and briquette can considerably
reduce the emission of toxic substances.

2.9 Processing of Ore and Coal Slurry

[0159] Furthermore, starch may be used as a flocculant in the processing
of ore and coal slurry.

2.10 Additive for Casting Materials

[0160] Another field of application is the use as an additive to process
materials in casting. For various casting processes cores produced from
sands mixed with binding agents are needed. Nowadays, the most commonly
used binding agent is bentonite mixed with modified starches, mostly
swelling starches.

[0161] The purpose of adding starch is increased flow resistance as well
as improved binding strength. Moreover, swelling starches may fulfil more
prerequisites for the production process, such as dispersability in cold
water, rehydratisability, good mixability in sand and high capability of
binding water.

2.11 Rubber Industry

[0162] In the rubber industry starch may be used for improving the
technical and optical quality. Reasons for this are improved surface
gloss, grip and appearance. For this purpose, starch is dispersed on the
sticky rubberised surfaces of rubber substances before the cold
vulcanization. It may also be used for improving the printability of
rubber.

2.12 Production of Leather Substitutes

[0163] Another field of application for modified starch is the production
of leather substitutes.

2.13 Starch in Synthetic Polymers

[0164] In the plastics market the following fields of application are
emerging: the integration of products derived from starch into the
processing process (starch is only a filler, there is no direct bond
between synthetic polymer and starch) or, alternatively, the integration
of products derived from starch into the production of polymers (starch
and polymer form a stable bond).

[0165] The use of the starch as a pure filler cannot compete with other
substances such as talcum. This situation is different when the specific
starch properties become effective and the property profile of the end
products is thus clearly changed. One example is the use of starch
products in the processing of thermoplastic materials, such as
polyethylene. Thereby, starch and the synthetic polymer are combined in a
ratio of 1:1 by means of coexpression to form a `master batch`, from
which various products are produced by means of common techniques using
granulated polyethylene. The integration of starch in polyethylene films
may cause an increased substance permeability in hollow bodies, improved
water vapor permeability, improved antistatic behaviour, improved
anti-block behaviour as well as improved printability with aqueous dyes.

[0166] Another possibility is the use of the starch in polyurethane foams.
Due to the adaptation of starch derivatives as well as due to the
optimisation of processing techniques, it is possible to specifically
control the reaction between synthetic polymers and the hydroxy groups of
the starch. The results are polyurethane films having the following
property profiles due to the use of starch: a reduced coefficient of
thermal expansion, decreased shrinking behaviour, improved
pressure/tension behaviour, increased water vapour permeability without a
change in water acceptance, reduced flammability and cracking density, no
drop off of combustible parts, no halides and reduced aging.
Disadvantages that presently still exist are reduced pressure and impact
strength.

[0167] Product development of film is not the only option. Also solid
plastics products, such as pots, plates and bowls can be produced by
means of a starch content of more than 50%. Furthermore, the
starch/polymer mixtures offer the advantage that they are much easier
biodegradable.

[0168] Furthermore, due to their extreme capability to bind water, starch
graft polymers have gained utmost importance. These are products having a
backbone of starch and a side lattice of a synthetic monomer grafted on
according to the principle of radical chain mechanism. The starch graft
polymers available nowadays are characterised by an improved binding and
retaining capability of up to 1000 g water per g starch at a high
viscosity. These super absorbers are used mainly in the hygiene field,
e.g. in products such as nappies and sheets, as well as in the
agricultural sector, e.g. in seed pellets.

[0169] What is decisive for the use of the novel starch modified by
recombinant DNA techniques are, on the one hand, structure, water
content, protein content, lipid content, fibre content, ashes/phosphate
content, amylose/amylopectin ratio, distribution of the relative molar
mass, branching degree, granule size and shape as well as
crystallization, and on the other hand, the properties resulting in the
following features: flow and sorption behaviour, pastification
temperature, viscosity, thickening performance, solubility, paste
structure, transparency, heat, shear and acid resistance, tendency to
retrogradation, capability of gel formation, resistance to
freezing/thawing, capability of complex formation, iodine binding, film
formation, adhesive strength, enzyme stability, digestibility and
reactivity.

[0170] The production of modified starch by genetically operating with a
transgenic plant may modify the properties of the starch obtained from
the plant in such a way as to render further modifications by means of
chemical or physical methods superfluous. On the other hand, the starches
modified by means of recombinant DNA techniques might be subjected to
further chemical modification, which will result in further improvement
of the quality for certain of the above-described fields of application.
These chemical modifications are principally known. These are
particularly modifications by means of

[0178] esterification [0179] leading to the formation of phosphate,
nitrate, sulfate, xanthate, acetate and citrate starches. Other organic
acids may also be used for the esterification.

[0180] In another embodiment, the present invention relates to parts of
plants of the invention that can be harvested, e.g. fruit, storage roots,
roots, blossoms, buds, sprouts or stems, preferably seeds or tubers with
said parts that can be harvested containing plants cells of the
invention.

[0181] In another aspect, the present invention relates to a regulatory
region which naturally controls, in bacterial cells, the transcription of
an above-described nucleic acid molecule of the invention encoding a
branching enzyme from bacteria of the genus Neisseria.

[0182] Within the meaning of the present invention, the term "regulatory
region" relates to a region that influences the specificity and/or the
extent of the expression of a gene sequence, e.g. in such a way that the
expression takes place in response to certain external stimuli or at a
certain time. Such regulatory regions usually are located in a region
that is called promoter. Within the meaning of the present invention, the
term "promoter" comprises nucleotide sequences that are necessary for
initiating the transcription, i.e. for binding the RNA polymerase, and
may also comprise the TATA box(es).

[0183] In a preferred embodiment, the regulatory region of the invention
comprises a nucleotide sequence selected from the group consisting of:
[0184] (a) nucleotide sequences comprising the nucleotides 1 to 169 of
the nucleotide sequence depicted in SEQ ID NO. 1; [0185] (b) the
nucleotide sequence of the regulatory region contained in the insert of
the plasmid DSM 12425 or parts thereof; and [0186] (c) nucleotide
sequences hybridizing with the sequences of (a) or (b) under stringent
conditions.

[0187] The nucleotides 1 to 169 of the sequence depicted in SEQ ID NO. 1
form part of the regulatory region of the gene of the branching enzyme
from Neisseria denitrificans. Putative promoter sequences are located at
the positions 36 to 44, 51 to 55 and 157 to 162, wherein the sequence
"GGGAGA" possibly is a Shine-Dalgarno sequence.

[0188] The present invention also relates to regulatory regions having a
homology to the aforementioned regulatory regions that is so high that
they hybridize to at least one of said regions, preferably under
stringent conditions. Regulatory regions that have a sequence identity of
at least 80%, preferably of at least 90% and most preferably of at least
95% to any of the aforementioned regulatory regions, in particular to the
one depicted in SEQ ID NO. 1, are particularly preferred.

[0189] They also comprise regulatory regions which are modified with
regard to the above-described regulatory regions, for instance due to
deletion(s), insertion(s), substitution(s), addition(s) and/or
recombination(s) and/or modification(s).

[0190] The skilled person is familiar with methods for introducing such
modifications into the regulatory regions. Moreover, the person skilled
in the art knows that the regulatory regions of the invention may be
coupled with further elements which influence the transcription in
bacterial cells, e.g. with enhancer elements.

[0191] The present invention also relates to recombinant DNA molecules
comprising a regulatory region of the invention.

[0192] In such a recombinant DNA molecule, the regulatory region is
preferred to be linked to a heterologous DNA sequence. In this context,
the term "heterologous" means that said sequence is naturally not linked
to the regulatory region. In addition, a recombinant DNA molecule of the
invention may contain further regulatory elements which are of importance
as regards transcription and/or translation in bacterial cells, e.g.
transcription or translation enhancers.

[0193] Moreover, the present invention relates to host cells that are
transformed with a regulatory region, a recombinant DNA molecule or a
vector of the invention.

[0194] Furthermore, the present invention relates to vectors containing a
regulatory region of the invention or a recombinant DNA molecule of the
invention. Said vectors comprise, for instance, also plasmids, cosmids,
bacteriophages, viruses, etc. which usually are used for methods in
molecular genetics.

[0195] In addition, the invention relates to an in-vitro method for
producing α-1,6-branched α-1,4-glucans using the substrate
sucrose and an enzyme combination of an amylosucrase and a branching
enzyme. Within the meaning of the present invention, the term "in-vitro
method" relates to a conversion, i.e. a reaction, which takes place
outside the living organism. In particular, the term "in vitro" means
that the method of the invention takes place in a reaction vessel. Most
preferably, the term "in vitro" means that the reaction takes place in
absence of living cells.

[0196] The advantage of the method of the invention is that it is possible
to control the branching degree and that it is possible, by means of said
control, to adapt the properties of the glucans synthesized to the
planned use of the glucans. Thus, as regards the application as
capsulation material in pharmaceutics, there is the possibility of
optimising the release rate of pharmaceutical agents by purposefully
adjusting the branching degree.

[0197] Within the meaning of the present invention, an amylosucrase
(sucrose:1,4-α-D-glucan 4-α-glucosyltransferase, E.C.
2.4.1.4) is an enzyme which catalyses the conversion of sucrose to
water-insoluble α-1,4-glucans and fructose. For said enzyme, the
following reaction scheme is suggested:

sucrose+(α-1,4-D-glucan)n→D-fructose+(α-1,4-D-gl-
ucan)n+1

[0198] This is a transglycosylation reaction. The products of said
reaction are water-insoluble α-1,4-glucans and fructose. The
transglycosylation may take place in the absence or in the presence of
acceptor molecules. Such acceptor molecules may be, for instance,
polysaccharides like malto-oligosaccharides, dextrin or glycogen. If said
acceptor molecule is a linear, oligomeric α-1,4-glucan, the product
resulting from the transglycosylation reaction by means of the
amylosucrase is a polymeric linear α-1,4-glucan. If the
transglycosylation reaction by means of amylosucrase is carried out
without any acceptor molecules, a glucan having a terminal fructose
molecule is obtained. Within the meaning of the present invention, all
products obtained by means of an amylosucrase in the absence or in the
presence of acceptor molecules are called α-1,4-glucans.

[0199] For the reaction mechanism of a transglycosylation by means of an
amylosucrase in the absence of an acceptor molecule, the following
reaction scheme is suggested:

G-F+n(G-F)→Gn-G-F+nF,

wherein G-F is sucrose, G is glucose, F is fructose and Gn-G-F is an
α-1,4-glucan.

[0200] For the reaction mechanism of a transglycosylation by means of
amylosucrase in the presence of an acceptor molecule, the following
reaction scheme is suggested:

mG-F+Gn→Gn-m+mF,

wherein Gn is a polysaccharide acceptor molecule, Gn-m is a
polysaccharide consisting of an acceptor plus an α-1,4-glucan chain
synthesized thereto, G-F is sucrose, F is fructose and G is glucose.

[0201] No co-factors are necessary for the transglycosylation by means of
an amylosucrase.

[0202] In principle, all amylosucrases which catalyse the synthesis of
linear α-1,4-glucans starting from sucrose are suitable for
carrying out the method of the invention.

[0203] Up to now, amylosucrases from several bacteria species have been
known, in particular mainly from Neisseria species (MacKenzie et al.,
Can. J. Microbiol. 24 (1978), 357-362).

[0205] In a preferred embodiment of the invention, an amylosucrase from
Neisseria polysaccharea is used.

[0206] The enzyme that is expressed in Neisseria polysaccharea is
extremely stable and binds very tight to the polymerization products and
is competitively inhibited by the reaction product fructose (MacKenzie et
al., Can. J. Microbiol. 23 (1977), 1303-1307). As regards the Neisseria
species Neisseria polysaccharea, the amylosucrase is secreted (Riou et
al., Can. J. Microbiol. 32 (1986), 909-911), whereas in other Neisseria
species, it remains in the cell. An amylosucrase having the amino acid
sequence depicted in SEQ ID NO. 5 is particularly preferred to be used.

[0207] In another preferred embodiment of the invention, a purified
amylosucrase is used.

[0208] In this context, a purified amylosucrase is an enzyme which is
substantially free of cellular components of the cells in which the
protein is synthesized. Preferably, the term "purified amylosucrase"
relates to an amylosucrase which has a degree of purity of at least 70%,
preferably of at least 85% and most preferably of at least 90%.

[0209] The use of a purified protein for producing α-1,4-glucans has
various advantages. In contrast to methods using partially purified
protein extracts, the reaction medium of the method of the invention does
not contain any residues of the production strain (microorganism) that is
used to purify the protein or to produce it by means of genetic
engineering.

[0210] What is more, there are advantages in the food and pharmaceutical
industries if the purified protein is used. The components of the product
are defined more exactly, too, if the reaction medium is defined and if
all unnecessary components have been removed. This leads to a less
extensive procedure for marketing authorisation for these products, which
have been manufactured by means of biotechnology, in the food and
pharmaceutical industry, in particular, since said products are supposed
to show no traces of a transgenic microorganism.

[0211] Within the meaning of the present invention, a branching enzyme
(α-1,4-glucan:α-1,4-glucan 6-glycosyltransferase, E.C.
2.4.1.18) is a protein catalysing a transglycosylation reaction in which
the α-1,4-linkings of an α-1,4-glucan donor are hydrolyzed
and the released α-1,4-glucan chains are transferred to an
α-1,4-glucan acceptor chain and converted into
α-1,6-linkings.

[0214] In a preferred embodiment of the invention, the branching enzyme is
a branching enzyme from a prokaryote, preferably from a bacterium of the
genus Neisseria, more preferably from Neisseria denitrificans and most
preferably from a branching enzyme of the invention as is described
below. A branching enzyme having the amino acid sequence depicted in SEQ
ID NO. 1 is particularly preferred.

[0215] In another preferred embodiment, the branching enzyme is a purified
branching enzyme. In this context, a purified branching enzyme is an
enzyme which is substantially free of cellular components of the cells in
which the protein is synthesized. Preferably, the term "purified
branching enzyme" means that the enzyme has a degree of purity of at
least 70%, preferably of at least 85% and most preferably of at least
90%.

[0216] Moreover, in the method of the invention, proteins are preferred to
be used which have been produced recombinantly. Within the meaning of the
present invention, said proteins are proteins which have been produced by
introducing a DNA sequence encoding said protein into a host cell and
expressing it there. The protein may subsequently be recovered from the
host cell and/or the culture medium. The host cell is preferred to be a
bacterium or a protist (e.g. fungi, in particular yeasts, algae), such as
defined, for example in Schlegel "Allgemeine Mikrobiologie" (Georg Thieme
Verlag, 1985, 1-2). In particular, the proteins are preferred to be
secreted by the host cell. Such host cells for producing a recombinant
protein can be generated using methods that are known to the person
skilled in the art.

[0217] Methods in Enzymology 153 (1987), 385-516, Bitter et al. (Methods
in Enzymology 153 (1987), 516-544; Sawers et al., Applied Microbiology
and Biotechnology 46 (1996), 1-9; Billmann-Jacobe, Current Opinion in
Biotechnology 7 (1996), 500-504; Hockney, Trends in Biotechnology 12
(1994), 456-463 and Griffiths et al., Methods in Molecular Biology 75
(1997), 427-440 give an overview of different expression systems.
Expression vectors have been described extensively in the literature.
Apart from a selection marker gene and a replication origin guaranteeing
the replication in the selected host, they usually contain a bacterial or
a viral promoter, and mostly a termination signal for the transcription.
Between the promoter and the termination signal, there is at least one
restriction site or a polylinker which allow the insertion of an encoding
DNA sequence. The DNA sequence which naturally controls the transcription
of the corresponding gene can be used as promoter sequence if it is
active in the selected host organism. Said sequence, however, may also be
exchanged for other promoter sequences. Both promoters effecting the
constitutive expression of the gene and inducible promoters allowing a
directed regulation of the expression of the downstream gene can be used.
Bacterial and viral promoter sequences having these properties have been
described extensively in the literature. Regulatory sequences for the
expression in microorganisms (e.g. E. coli, S. cerevisiae) have been
described sufficiently in the literature. Promoters allowing a
particularly strong expression of the downstream gene include, for
example, the T7 promoter (Studier et al., Methods in Enzymology 185
(1990), 60-89), lacuv5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and
Chamberlin (eds.) Promoters, Structure and Function; Praeger, New York
(1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983),
21-25), p 1, rac (Boros et al., Gene 42 (1986), 97-100). Normally, the
amounts of proteins reach their top level from the middle to about the
end of the logarithmic phase of the growth cycle of the microorganisms.
Therefore, preferably inducible promoters are used for the synthesis of
proteins. These inducible promoters often result in a higher yield of
proteins than the constitutive promoters. Due to the constant
transcription and translation of a cloned gene, the use of strong
constitutive promoters often has the effect that the energy for other
essential cell functions is lost and that, thus, the cell growth is
slowed down (Bernard R. Glick/Jack J. Pasternak, Molekulare
Biotechnologie (1995), Spektrum Akademischer Verlag GmbH, Heidelberg
Berlin Oxford, p. 342). Hence, a two-step method is often used to achieve
the optimum amount of proteins. First, the host cells are cultivated
under optimum conditions until they reach a relatively high cell density.
In the second step, the transcription is induced depending on the kind of
promoter used. In this context, a tac promoter that is inducible by
lactose or IPTG (=isopropyl-β-D-thiogalacto-pyranoside) is
particularly suitable (DeBoer et al., Proc. Natl. Acad. Sci. USA 80
(1983), 21-25). Termination signals for the transcription have also been
described in the literature.

[0218] The transformation of the host cell with the DNA encoding a
corresponding protein DNA can normally be carried out according to
standard methods, as described, for instance, in Sambrook et al.
(Molecular Cloning: A Laboratory Course Manual, 2nd edition (1989),
Cold Spring Harbor Press, New York). The host cell is cultivated in
culture media which correspond to the needs of the respective host cell.
In particular, pH value, temperature, salt concentration, aeration,
antibiotics, vitamins and trace elements, etc. are taken into
consideration.

[0219] The enzyme produced by the host cells can be purified according to
standard purification techniques, such as precipitation, ion exchange
chromatography, affinity chromatography, gel filtration, HPLC reverse
phase chromatography, etc.

[0220] By modifying the DNA expressed in the host cells, it is possible to
produce a polypeptide in the host cell, which is easier to be isolated
from the culture medium due to certain properties. Thus, there is the
possibility of expressing the protein to be expressed as a fusion protein
together with another polypeptide sequence the specific binding property
of which allows the isolation of the fusion protein through affinity
chromatography (e.g. Hopp et al., Bio/Technology 6 (1988), 1204-1210;
Sassenfeld, Trends Biotechnol. 8 (1990), 88-93).

[0221] In a preferred embodiment of the method of the invention, enzymes
are used which have been produced recombinantly and which have been
secreted by the host cell into the culture medium so that it is not
necessary to disrupt cells or to purify the protein any further since the
secreted protein may be recovered from the supernatant. Methods known in
process engineering, such as dialysis, reverse osmosis, chromatographic
methods, etc. may be used for removing residual components of the culture
medium. The same applies to the reconcentration of the protein secreted
into the culture medium. Normally, the secretion of proteins by
microorganisms is mediated by N-terminal signal peptides (signal
sequence, leader peptide). Proteins having said signal sequence may pass
through the cell membrane of the microorganism. Secretion of proteins may
be achieved by linking the DNA sequence that encodes said signal peptide
to the corresponding region encoding the enzyme.

[0222] A signal peptide that optionally occurs naturally is preferred,
e.g. the signal peptide of the amylosucrase from Neisseria polysaccharea.

[0223] The signal peptide of the α-CGTase from Klebsiella oxytoca
M5A1 (Fiedler et al., J. Mol. Biol. 256 (1996), 279-291) or a signal
peptide as is encoded by the nucleotides 11529-11618 of the sequence
accessible in the GenBank under the accession number X86014 is
particularly preferred.

[0224] As an alternative, the enzymes used in the method of the invention
may also have been produced using an in-vitro transcription and
translation system which leads to the expression of the proteins without
using microorganisms.

[0225] In another preferred embodiment, the amylosucrase and/or the
branching enzyme are immobilized on a support material.

[0226] Immobilizing the enzymes has the advantage that the enzymes can be
recovered from the reaction mixture in a simple manner as catalysts of
the synthesis reaction and can be used several times. Since the
purification of enzymes usually requires much time and money,
immobilization and recycling can save costs considerably. The degree of
purity of the reaction products which do not contain any remaining
proteins is another advantage.

[0227] There is a plurality of support materials at disposal for
immobilizing proteins wherein the coupling with the support material may
take place via covalent or non-covalent bindings (for an overview see:
Methods in Enzymology 135, 136, 137). For example, agarose, alginate,
cellulose, polyacrylamide, silica or nylon are extensively used as
support material.

[0228] In another preferred embodiment of the method, a (partially
purified) enzyme crude extract of an amylosucrase and/or a branching
enzyme is used. In this context, a crude extract is an amylosucrase
and/or branching enzyme preparation having a reduced degree of purity in
comparison with a purified enzyme (cf. Examples 5 and 6).

[0229] In a preferred embodiment, in the method of the invention the
branching degree of the α1,6-branched α-1,4-glucans is
modified by changing the ratio of the protein activity of branching
enzyme and amylosucrase. In this context, the ratio of the protein
activity is the ratio of the protein activities (u) from amylosucrase and
branching enzyme. The protein activities may be determined as described
in Examples 7 and 8. When the method of the invention is carried out (cf.
Example 9), the ratio of protein activity (units of amylosucrase/units of
branching enzyme) may range from 1/4000 to 2000/1.

[0230] In a preferred embodiment, the ratio of the protein activity ranges
from 1/1500 to 1500/1.

[0231] In another preferred embodiment, the ratio of the protein activity
ranges from 1/800 to 1300/1.

[0232] In a particularly preferred embodiment, the ratio of the protein
activity ranges from 1/400 to 1200/1.

[0233] It is possible to modify the branching degree of the
α-1,6-branched α-1,4-glucans obtained from 0.05% to 35% by
changing the ratio of the protein activity.

[0234] In a preferred embodiment, it is possible to change the branching
degree of the α-1,6-branched α-1,4-glucans in 6-position from
0.15% to 25%, more preferably from 0.20% to 15% and most preferably from
0.25% to 12%.

[0235] If the method of the invention is used, it is possible, in
particular, to produce products having a higher branching degree than
glycogen.

[0236] Within the meaning of the present invention, the branching degree
is the average share of branchings in O-6 position compared to all
glucose units linked differently. The branching degree can be determined
by methylation analysis (cf. Example 10).

[0237] In another preferred embodiment, in the method of the invention,
the molecular weight of the products is modified by changing the protein
activity ratio. It is, in particular, possible to change the protein
activity ratio during the reaction that leads to the synthesis of the
α-1,6-branched α-1,4-glucans.

[0238] In another preferred embodiment of the method of the invention, the
method is to be carried out at different sucrose concentrations. In
principle, it is possible for the method to be carried out at a
concentration preferably ranging from 1% to 80% sucrose (w/v), more
preferably ranging from 5% to 50% and most preferably from 10% to 40%.

[0239] In the present invention, the molecular weight is determined by
light scattering experiments (Light Scattering from Polymer Solutions,
editor: Huglin, M. B., Academic Press, London, 1972) according to Berry
(J. Chem. Phys. 44 (1966), pp. 4550). By means of the method of the
invention, it is possible, in particular, to adjust the molecular weight
of the α-1,6-branched α-1,4-glucans produced by said method
to a range of 1000 to 3000×106. Preferably, the
α-1,6-branched α-1,4-glucans have a molecular weight ranging
from 100,000 to 1500×106, more preferably from 100,000 to
1000×106, even more preferably from 262,000 to
1000×106 and most preferably from 262,000 to
499×106.

[0240] Furthermore, the invention relates to α-1,6-branched
α-1,4-glucans obtainable by the above-described method of the
invention. Said α-1,6-branched α-1,4-glucans have a branching
degree which is higher than the one that is achieved if only the activity
of an amylosucrase is used and which is 25 mol % at the most.

[0241] In a preferred embodiment of the invention, these are
α-1,6-branched α-1,4-glucans having a branching degree
ranging from 0.05% to 20%, preferably from 0.15% to 17%, more preferably
from 0.2% to 15%, even more preferably from 0.25% to 13% and most
preferably from 0.3% to 12%. In another preferred embodiment of the
invention, the branching degree ranges from 0.35% to 11% and, in
particular, from 0.4% to 10.5%.

[0242] The α-1,6-branched α-1,4-glucans of the invention can
be used in the food and non-food industries as has been described above
with regard to the starch of the invention.

[0243] The plasmid pBB48, which has been produced within the present
invention, was deposited with the Deutsche Sammlung von Mikroorganismen
and Zellkulturen (DSMZ, German Collection of microorganisms and cell
cultures) in Braunschweig, which is approved as international depository,
on 25 Sep. 1998 with the accession number DSM 12425 according to the
requirements of the Budapest Treaty.

[0245] FIG. 2 shows a number of α-1,4-glucans having a varying
degree of α-1,6-branchings which were produced by means of the
method of the invention and which were subsequently dyed with Lugol's
solution.

[0247] FIG. 3 shows a HPLC chromatograph of a highly branched process
product (A) which has been debranched with isoamylase and a rat liver
glycogen sample (B) which has been debranched with isoamylase.

[0248] FIG. 4 shows the scheme of the methylation analysis.

[0249] FIG. 5 shows a diagram of the results of the analysis of sample 7
described in Examples 9 and 10 after one and after two methylation steps.
The values for the 2,3,6-methylation are 96.12% and 96.36%, respectively.

[0251] FIGS. 7 and 8 show gas chromatographs of the samples 3 and 7
described in the Examples.

[0252] FIG. 9 schematically shows the plasmid pBE-fnr-Km.

[0253] FIG. 10 shows an activity gel for the branching enzyme.

[0254] FIG. 11 shows the schematic illustration of an RVA profile.

[0255] FIG. 12 shows the distribution of granule size of the lines 143-13A
and 143-59A compared to the wild type.

[0256] FIG. 13 shows the microscopic magnification of the starch granules
of the lines 143-13A, 143-34A and 143-59A in comparison with the starch
granules of wild type plants (light microscope by Leitz, Germany).

[0257] FIG. 14 shows the gel texture of the starches of different
transgenic lines compared to starches from wild type plants. The texture
was determined by means of a texture analyzer.

[0258] FIG. 15 shows the RVA profile of the starches of the lines 143-11A,
143-13A, 143-59A compared to the wild type.

[0259] FIGS. 16 to 18 show the results of HPLC chromatographies which
represent the pattern of the distribution of the side-chains of the lines
143-WT (=wild type), 143-13A and 143-59A.

[0260] FIG. 19 shows the elution gradient that was used for the
chromatographies depicted in FIGS. 16 to 18.

[0261] FIG. 20 shows the percentage deviation of side-chains having
certain chain lengths of the starches analysed in FIGS. 16 to 18 from the
wild type.

[0267] Starch was isolated from potato plants according to standard
methods and the ratio of amylose to amylopectin was determined according
to the method described by Hovenkamp-Hermelink et al. (Potato Research 31
(1988), 241-246).

(b) Determination of the Phosphate Content

[0268] In starch, the positions C2, C3 and C6 of the glucose units may be
phosphorylated.

[0270] The overall content of phosphate was determined according to the
method by Ames (Methods in Enzymology VIII (1966), 115-118).

[0271] Approximately 50 mg starch are added to 30 μl of an ethanolic
magnesium nitrate solution and ashed for 3 hours at 500° C. in a
muffle furnace. 300 μl 0.5 M hydrochloric acid were added to the
residue and incubated for 30 min at 60° C. Then, an aliquot is
filled up to 300 μl 0.5 M hydrochloric acid, added to a mixture of 100
μl of 10% ascorbic acid and 600 μl of 0.42% ammonium molybdate in 2
M sulphuric acid and incubated for 20 min at 45° C.

[0272] Then, a photometric determination at 820 nm is carried out with a
calibration curve using phosphate standards.

(c) Determination of the Gel Texture (Texture Analyzer)

[0273] 2 g starch (TS) are pasted in 25 ml H2O (cf. RVA) and
subsequently sealed airtight and stored at 25° C. for 24 hours.
The samples are fixed under the probe (round stamp) of a texture analyzer
TA-XT2 by Stable Micro Systems and the gel texture is determined with
regard to the following parameters:

[0274] 2 g starch (TS) are added to 25 ml H2O and put in a Rapid
Visco Analyzer (Newport Scientific Pty Ltd., Investment Support Group,
Warriewod NSW 2102, Australia) for analysis. The device was operated
according to the manufacturer's instructions. For determining the
viscosity of the aqueous solution of the starch, first of all, the starch
suspension is heated from 50° C. to 95° C. at a speed of
12° C. per minute. Then, the temperature is maintained for 2.5
minutes at 95° C. Subsequently, the solution is cooled down from
95° C. to 50° C. at a speed of 12° C. per minute.
The viscosity is determined during the whole time.

[0275] The pastification temperature is determined by means of the slope
of the viscosity graph depending on the time. If the slope of the graph
is higher than 1.2 (this value is set by the user), the computer
programidentifies the temperature measured in this moment as
pastification temperature.

(e) Determination of Glucose, Fructose and Sucrose

[0276] The content of glucose, fructose and sucrose is determined
according to the method described by Stitt et al. (Methods in Enzymology
174 (1989), 518-552).

(f) Analysis of the Distribution of the Side-Chains of the Amylopectin

[0277] The distribution of the side-chains and the preparation are
determined as described in Lloyd et al. (Biochem. J. 338 (1999),
515-521). It is pointed to the fact that, using said method, only the
amylopectin is debranched and that the amylose is separated from the
amylopectin before debranching by means of thymol precipitation. The
following conditions for the elution are selected (simplified
illustration, the exact elution profile is shown in FIG. 19):

[0278] The size of the granules was determined with a photosedimentometer
of the type "Lumosed" by Retsch GmbH, Germany.

[0279] The distribution of the granule size was determined in an aqueous
solution and was carried out according to the manufacturer's indications
as well as on the basis of the literature, e.g. H. Pitsch,
Korngroβenbestimmung; LABO-1988/3 Fachzeitschrift fur Labortechnik,
Darmstadt.

(h) Determination of the Water-Binding Capacity

[0280] For determining the water-binding capacity, the residue was weighed
after separating the soluble parts of the starch swelled at 70° C.
by means of centrifugation. The water-binding capacity (WBV) of the
starch was determined with reference to the initial weight that was
corrected by the soluble mass.

Isolation of a Genomic DNA Sequence Encoding a Branching Enzyme from
Neisseria denitrificans

[0281] For isolating the branching enzyme from Neisseria denitrificans,
first of all, a genomic library was established. For this purpose, cells
of Neisseria denitrificans of the strain deposited as ATCC 14686 at the
ATCC were cultivated on Columbia blood agar plates and subsequently
harvested. The genomic DNA was isolated and purified according to the
method by Ausubel et al. (in: Current Protocols in Molecular Biology
(1987); J. Wiley & Sons, NY). After a partial restriction digestion with
the restriction endonuclease Sau3A, a ligation with BamHI-cleaved phage
vector DNA (lambdaZAPExpress by Stratagene) was carried out. After the
in-vivo excision of the phage library, the plasmids obtained were
transformed into the E. coli mutant (PGM-) (Adhya and Schwartz, J.
Bacteriol. 108 (1971), 621-626). When growing on maltose, said mutant
forms linear polysaccharides which turn blue after colouring with iodine.
60,000 transformants were plated onto YT agar plates with IPTG (1 mM),
kanamycin (12.5 mg/l) and maltose (1%) and after incubation for 16 hours
at 37° C., they were vaporized with iodine. 60 bacteria colonies
which had a red, brown or yellow colour after vaporization with iodine
were sleeted and plasmid DNA was isolated therefrom (Birnboim-Doly,
Nucleic Acid Res. 7, 1513-1523). The isolated plasmids were then used for
retransformation of the same E. coli-(PGM)-mutant (Adhya and Schwartz, J.
Bacteriol. 108 (1971), 621-626). After repeated plating and vaporization
with iodine, the clones could be reduced from 60 isolates to 4 isolates.
A restriction analysis was carried out with these four plasmids showing
an EcoRI fragment (1.6 kb) which had the same size in all four plasmids
(FIG. 1).

EXAMPLE 2

Sequence Analysis of the Genomic Fragment of the Plasmid pBB48

[0282] The 1.6 kb EcoRI fragment was isolated (Geneclean, Bio101) from a
clone obtained according to Example 1 (pBB48) which had an approx. 3.9 kb
insert in the vector pBK-CMV (Stratagene). For DNA sequencing, the
fragment was cloned into the vector pBluescript which had been cleaved
with EcoRI. The plasmid obtained in this way was sequenced. Then, the
entire DNA sequence encoding the branching enzyme as well as the sequence
of flanking regions was determined by means of the starting plasmid pBB48
(SEQ ID NO. 1). The plasmid pBB48 is shown in FIG. 1. The plasmid is
deposited under DSM 12425.

EXAMPLE 3

Expression of the Branching Enzyme in Recombinant E. coli Cells

[0283] In general, an endogenous branching enzyme (glgB) is expressed in
the E. coli laboratory strains. For this reason, the G6MD2 mutant of E.
coli was used for detecting the branching enzyme activity. The strain E.
coli Hfr G6MD2 (E. coli Genetic Stock Center, Yale University, CGSC#5080)
has an extended deletion in the region of the glucan synthesis genes
(glgA, glgB, glgC). For detecting the branching enzyme activity, said
mutant was transformed with the plasmid pBB48 and a crude extract was
prepared of the propagated cells. The proteins of said crude extract were
separated electrophoretically in a polyacrylamide gel and then incubated
with and without rabbit phosphorylase B (100 mM sodium citrate, pH 7.0;
AMP, glucose-1-phosphate) for determining the branching enzyme activity.
Violet bands only appeared in the gel stimulated with phosphorylase,
which indicated a strong branching enzyme activity.

EXAMPLE 4

[0284] In-Vitro Production of α-1,6-Branched α-1,4-Glucans
with Protein Crude Extracts in a Cell-Free System

[0285] For the expression of the branching enzyme, the mutant E. coli
G6MD2 was transformed with the plasmid pBB48. The cells were cultivated
with YT medium with kanamycin (12.5 mg/l) for 16 hours while shaking in
an Erlenmeyer flask. After centrifugation (5000×g), the pellet
obtained was washed with 100 mM Tris/HCl, pH 7.5, 1 mM DTT and, after
suspension in the same buffer, the cells were disrupted with an
ultrasonic probe. By another centrifugation (10,000×g), the cell
debris was separated from the soluble proteins and a yellowish
supernatant having a protein concentration of approx. 10 mg/ml was
obtained.

[0286] From the protein crude extract obtained in that manner, different
amounts (100 μl, 10 μl, 1 μl, 0.1 μl, 0.01 μl, 0.001
μl) were added to an unchanged amount of an amylosucrase in 50 ml 100
mM sodium citrate, pH 7.0 with 20% sucrose and 0.02% sodium azide. After
a few hours, a first clouding was observed in the reaction mixture. After
three days, the mixture was centrifuged and the products formed were
washed with deionized water.

[0287] The products are soluble in DMSO and may be characterised by
measuring an absorption spectrum with Lugol's solution by means of which
the branching degree of the products formed may be estimated. For this
purpose, the DMSO solution was strongly diluted with water and Lugol's
solution was added and the spectrum from 400 nm to 700 nm was immediately
measured in a Beckmann spectrophotometer (cf. FIG. 2).

[0288] Separation of the side-chains that were split off with isoamylase
on a Carbopak PA100 column by means of HPLC (DIONEX; running agent: 150
mM NaOH with 1 M sodium acetate gradient) shows the same pattern for a
strongly branched product as for a rat liver glycogen debranched with
isoamylase (FIG. 3).

[0289] After incubation with a pullulanase, the side-chains were only
split off to a very small extent.

EXAMPLE 5

Purification of the Branching Enzyme and N-Terminal Sequencing of the
Protein

[0290] For isolating the branching enzyme of Neisseria denitrificans from
recombinant Hfr G6MD2 E. coli cells (see above), which had been
transformed with pBB48, first an overnight culture of said cells was
centrifuged. The cell precipitate was then suspended in 3 volumes
disruption buffer and disrupted in the French press at a pressure of
approx. 16,000 to 17,000 psi. After centrifugation at 10,000 g for one
hour, the supernatant was diluted to reach the 4-fold volume by adding
washing buffer. Then, it was bound to DEAE cellulose DE52 using the
batch-method and filled into a chromatography column which was washed
with 2 to 3 column volumes of washing buffer. Subsequently, a linear 1 M
NaCl gradient was applied for elution. The fractions with branching
enzyme activity were combined (see Example 8), (NH4)2SO4
was added (final concentration 20% (w/v)) and applied to a TSK butyl
Toyopearl 650M column. After washing with 2 to 3 column volumes of HIC
buffer, to which additionally an ammonium sulphate solution with a degree
of saturation of 20% (114 g ammonium sulphate per litre) had been added
before, the branching enzyme was eluted in HIC buffer using an ammonium
sulphate gradient that falls linearly from 20% to 0%. Fractions with
branching enzyme activity were combined. For concentrating the protein,
the purification step with the combined fractions was subsequently
repeated using a small TSK butyl Toyopearl 650M column (Tose Haas
(Montgomery VIIIe, Pennsylvania)). The purified protein was then applied
to a polyacrylamide gel, blotted onto a PVDF membrane, dissolved again
and sequenced N-terminally by WITA GmbH, Teltow, Germany, according to
the Edman method. The sequence obtained was: MNRNXH (SEQ ID NO. 3).

EXAMPLE 6

Purification of an Amylosucrase

[0291] For producing an amylosucrase, E. coli cells were used which had
been transformed with a DNA encoding an amylosucrase from Neisseria
polysaccharea. The DNA has the nucleotide sequence depicted in SEQ ID NO.
4 and is derived from a genomic library of N. polysaccharea.

[0292] An overnight culture of said E. coli cells which secrete the
amylosucrase from Neisseria polysaccharea was centrifuged off and
resuspended in approx. 1/20 volume of 50 mM sodium citrate buffer (pH
6.5), 10 mM DTT (dithiothreitol), 1 mM PMSF
(phenylmethylsulfonylfluoride). Then, the cells were disrupted twice with
a French press at 16,000 psi. Subsequently, 1 mM MgC12 and benzonase (by
Merck; 100,000 units; 250 units μl-1) were added to the cell
extract in a final concentration of 12.5 units ml-1. After that, the
mixture was incubated at 37° C. for at least 30 min while shaking
gently. The extract was left to stand on ice for at least 1.5 hours.
Then, it was centrifuged at 4° C. for 30 min at approx. 40,000 g
until the supernatant was relatively clear.

[0293] A pre-filtration with a PVDF membrane (Millipore "Durapore", or
similar) was carried out which had a pore diameter of 0.45 μm. The
extract was left to stand over night at 4° C. Before carrying out
the HI-(hydrophobic interaction) chromatography, solid NaCl was added to
the extract and adjusted to a concentration of 2 M NaCl. Then, the
mixture was again centrifuged at 4° C. for 30 min at approx.
40,000 mg. Subsequently, the remaining residues of E. coli were removed
from the extract by filtering it with a PVDF membrane (Millipore
"Durapore" of similar) which had a pore diameter of 0.22 μm. The
filtered extract was separated on a butylsepharose-4B column (Pharmacia)
(volume of the column: 93 ml, length: 17.5 cm). Approx. 50 ml of the
extract having an amylose activity of 1 to 5 units μl-1 were
applied to the column. Then, non-binding proteins were washed off the
column with 150 ml buffer B (buffer B: 50 mM sodium citrate, pH 6.5, 2 M
NaCl). Finally, the amylosucrase was eluted by means of a falling linear
NaCl gradient (from 2 M to 0 M NaCl in 50 mM sodium citrate in a volume
of 433 ml at an influx rate of 1.5 ml min-1) which had been
generated by means of an automatic pumping system (FPLC, Pharmacia). The
elution of the amylosucrase occurred between 0.7 M and 0.1 M NaCl. The
fractions were collected, desalted on a PD10 sephadex column (Pharmacia),
stabilised with 8.7% glycerol, examined for amylose sucrose activity and
finally deep-frozen in storage buffer (8.7% glycerol, 50 mM citrate).

EXAMPLE 7

Determination of the Amylosucrase Activity

[0294] The amylosucrase activity was determined by incubating purified
protein or protein crude extract in different dilutions at 37° C.
in 1 ml reaction mixtures containing 5% sucrose, 0.1% dextrin and 100 mM
citrate, pH 6.5. After 0 min, 30 min, 60 min, 120 min, 180 min, 240 min,
300 min and 360 min, 10 μl each are taken from said mixture, and the
enzymatic activity of the amylosucrase is stopped by immediate heating to
95° C. Then, the proportion of the fructose released by the
amylosucrase is determined in a combined photometric test. 1 μl to 10
μl of the inactivated sample are put in 1 ml 50 mM imidazole buffer,
pH 6.9, 2 mM MgCl2, 1 mM ATP, 0.4 mM NAD.sub.+ and 0.5 U/ml
hexokinase. After sequential addition of glucose-6-phosphate
dehydrogenase (from Leuconostoc mesenteroides) and phosphoglucose
isomerase, the change in the absorption is measured at 340 nm.
Subsequently, the amount of fructose released is calculated by means of
the Lambert-Beer law.

[0295] If the value obtained is brought into relation with the time when
the sample is taken, the number of units (1 U=μmol fructose/min) (per
μl protein extract or μg purified protein) can be determined.

EXAMPLE 8

Determination of the Enzyme Activity of a Branching Enzyme from Neisseria
denitrificans

[0296] The enzymatic activity of the branching enzyme was determined in
accordance with a method described in the literature (Krisman et al.,
Analytical Biochemistry 147 (1985), 491-496; Brown and Brown, Meth.
Enzymol. 8 (1966), 395-403). The method is based on the principle of
reduced iodine binding-affinity of branched glucans in comparison with
non-branched α-1,4-glucans.

[0297] For determining the enzymatic activity of the branching enzyme, a
series of samples of various dilutions of the branching enzyme was put
into a cooled micro-titre plate. Then, the reaction was started by adding
190 μl of an amylose reaction mixture (preparation see below) and
incubated at 37° C. in an incubator. Exactly after 30 min, the
reaction was stopped by adding 100 μl of Lugol's solution (0.5 mM) and
the samples were measured in a micro-titre reading device (Molecular
Devices) at 650 nm. A mixture without amylose served as control. The
reference sample with the maximum extinction value which contained
amylose but no branching enzyme had an OD650 of 1.2.

[0298] In order to be able to better compare independent assays, only the
sample dilution is used for the calculation which leads to a decrease of
the OD650 by 0.5 units during an incubation time of 30 min

Definition of an Activity Unit (U) of the Branching Enzyme:

[0299] The amount of enzymes causing a decrease of the OD650 by 0.5
units from 1.2 to 0.7 in 30 min in the test described is half a unit of
the branching enzyme.

Preparation of the Amylose Reaction Mixture:

[0300] While stirring, 1 ml of a 0.5% amylose solution (manufacturer:
Fluka; amylose from potato) w/v in DMSO are added to 10 ml sodium citrate
buffer (100 mM, pH 6,5, 0.02% w/v NaN3). For measuring, the clear
stock solution is again diluted with sodium citrate buffer to a ratio of
1:4 to 1:8. In the test, absorption with Lugol's solution should be at
1.2 in the reference sample used (maximum).

EXAMPLE 9

Production of α-1,6-Branched α-1,4-Glucans Having Different
Branching Degrees

[0301] For producing α-1,6-branched α-1,4 glucans having
different branching degrees, purified amylosucrase from Neisseria
polysaccharea (cf. Example 6) and a purified branching enzyme from
Neisseria denitrificans (cf. Example 5) were added to a 20% sucrose
solution (w/v) in a reaction volume of 10.86 ml. Depending on the test
mixture, the two enzymes were used in different protein activity ratios
to each other (for the determination amylosucrase see Example 7; for the
determination of the branching enzyme see Example 8) (see Table 1):

Determination of the Branching Degree by Means of Methylation Analysis

[0302] The branching degree of the glucans obtained was subsequently
determined by means of a methylation analysis.

1. Examinations Carried Out

[0303] methylation of all free OH-groups of the glucan samples, each
time double determination [0304] hydrolysis of the permethylated polymers
followed by a reduction at C-1 and acetylation of the monomer mixture
[0305] gas chromatographic analysis and quantification of the reaction
products

[0306] The branching degree of the glucan samples was established by means
of a methylation analysis (cf. FIG. 4). The free OH-groups of the polymer
are labelled by conversion into methylether.

[0307] The degradation to monomers is carried out in an acid hydrolytic
manner and leads to partially methylated glucose molecules which are
present in pyranosidic/furanosidic form and as α- and
α-glucosides. These variants are focussed by reduction with
NaBH4 in the corresponding partially methylated sorbite derivative.
By subsequent acetylation of free OH-groups the reaction products can be
examined by means of gas chromatography.

[0308] The following table shows the texture and the DMSO solubility of
the glucans obtained.

[0310] 1% solutions (w/v) were prepared in DMSO. Not all of the samples
were well-soluble at room temperature: 1, 3 and 13 had to be heated for
30 minutes to 110° C. Apart from the solutions 1 and 3, which were
slightly cloudy, there were optically clear solutions (cf. Table 2).

[0311] b) Methylation

[0312] 2 ml of the DMSO solution (i.e. 20 mg polymer) were transferred to
a 50 ml-nitrogen flask, added to 5 equivalents/OH (eq/OH) of freshly
prepared dimsyl solution in an N2 stream and stirred for 30 minutes. The
solutions turned cloudy and viscous. The content of the flask was frozen
in an ice-bath, 10 eq/OH methyliodide were added and, after thawing, the
mixture was stirred for at least 2 hours. Before the second deprotonation
and methylation step, surplus methyliodide was removed in the vacuum.

[0313] After removing the surplus methyliodide, processing was carried out
by adding 50 ml water and after extracting 5 times with 10 ml
dichloromethane each. Any traces of DMSO were removed from the organic
phase by extracting 3 times with water, then the organic phase was dried
with CaCl2, filtered and concentrated. The products were clear,
yellowish films.

[0314] By means of sample 7, it was first checked how many methylation
steps are necessary for the permethylation of the hydroxyl groups. After
the first methylation, half of the mixture was processed, the other half
was methylated again. After both samples had been degraded, the results
of the GC-analyses were compared. First, it was found that the reaction
had almost been quantitatively after one methylation step (cf. FIG. 5).
For identifying a possible branching at C-3, which also may only seem to
be present due to submethylation at said position, a second methylation
was carried out in any case.

[0315] FIG. 5 shows a diagram of the results of the analysis of sample 7
after one and after two methylation steps; the values for
2,3,6-methylation are 96.12% and 96.36%, respectively.

[0316] c) Hydrolysis

[0317] 2 mg of the methylated sample were weighed-in in a 1 ml-pressure
glass, 0.9 ml 2 M trifluor acetic acid were added and it was stirred for
2.5 hours at 120° C. After cooling the glass, the mixture was
concentrated in an Na stream. For removing traces of acid, three times
toluene was added and blown off.

[0319] 0.5 ml of an 0.5 M ammoniacal NaBD4 solution was added to the
remainder of the previous reaction step and stirred for 1 hour at
60° C. The reagent was carefully destroyed with a few drops of
glacial acetic acid. The resulting borate was removed by adding five
times a 15% methanolic acetic acid and subsequently blowing off as boric
acid trimethylester.

[0320] e) Acetylation

[0321] 50 μl pyridine and 250 μl acetic acid anhydride was added to
the remainder of the previous reaction step and stirred for 2 hours at
95° C. After cooling, the reacting mixture was dripped into 10 ml
saturated NaHCO3 solution and extracted five times with
dichloromethane. The reaction products in the organic phase were examined
by means of gas chromatography (product, cf. FIG. 4).

[0322] f) Gas Chromatography

[0323] The examinations by means of gas chromatography were carried out
using a device by Carlo Erby GC 6000 Vega Series 2 with on-column inlet
and FID detector. The separations were conducted on a fused-silica
capillary column called Supelco SPB5 (inner diameter 0.2 mm, length 30 m)
using hydrogen as carrier gas and a pressure of 80 kPa. The following
temperature programme was used: 60° C. (1 min)-25°
C./min→130° C.-4° C./min→280° C.

3. Results

[0324] The gas chromatographs were analysed by identifying the peaks,
integrating the peak areas and correcting the data by means of the ECR
concept by Sweet et al. (Sweet et al., Carbohydr. Res. 40 (1975), 217).

[0325] The 1,6-anhydro-compounds that could be observed in samples 1 and 3
are due to the high branching degree at C-6. During hydrolysis, this
leads to monomers having a free OH-group at C-6 which may further react
to form these derivatives under the reaction conditions. When calculating
the branching degree, these proportions have to be added to the "2,3-Me"
value.

[0326] FIG. 6 is an illustration of the proportions of terminal ("2346Me")
and b-linked ("23"Me) glucose units of the glucan samples examined.

Production of α-1,6-Branched α-1,4-Glucans Having Different
Molecular Weights

[0327] For producing α-1,6-branched α-1,4-glucans having
different molecular weights, a purified amylosucrase from Neisseria
polysaccharea (cf. Example 6) and a purified branching enzyme from
Neisseria denitrificans (cf. Example 5) were added to a 20% sucrose
solution (w/v) in a reaction volume of 10.86 ml. Depending on the test
mixture, the two enzymes were used in different protein activity ratios
(for the determination of the amylosucrase activity see Example 7; for
the branching enzyme see Example 8) (cf. Table 1). The molecular weights
and the radius of inertness Rg were determined by means of light
scattering (Light Scattering from Polymer Solutions; editor: Huglin, M.
B., Academic Press, London, 1972). The dried samples 1-11 were dissolved
in DMSO, H2O (at a ratio of 90:10) and different dilutions (approx.
2.5 g/l to 0.25 g/l) were analysed in a device for measuring the light
scattering (SOFICA, Societe francaise d'instruments de controle et
d'analyses. Le Mesnil Saint-Denis, France). The data obtained in this way
were [ . . . ]1 according to Berry (J. Chem. Phys. 44 (1966), 4550
et seq.). 1translator's note: verb missing.

[0328] Construction of an Expression Cassette for Transforming Plants for
the Plastidial Expression of a Branching Enzyme from Neisseria
denitrificans

[0329] The oligonucleotides BE-5' and BE-3' (SEQ ID NO. 6 and SEQ ID NO.
7) were used for amplifying the sequence coding for the branching enzyme
from Neisseria denitrificans by means of PCR starting from the plasmid
pBB48 (deposited with the Deutsche Sammlung von Mikroorganismen and
Zellkulturen (DSMZ, German Collection of microorganisms and cell
cultures) in Braunschweig with the accession number DSM 12425). The
resulting amplified sequences therefrom were digested with the
restriction endonucleases SalI and SdaI and cloned into the plasmid
pBinAR-fnr which was cleaved with SalI and SdaI. The plasmid resulting
therefrom was denoted pBE-fnr-Km (FIG. 9).

[0332] By means of Northern blot analysis, it was possible to identify
from the transgenic potato plants produced according to Example 12 plants
which displayed an mRNA of a branching enzyme from Neisseria
denitrificans. For detecting the activity of the branching enzyme in the
stably transformed plants, leaf material of the plants to be examined was
deep-frozen in liquid nitrogen and then ground in a mortar pre-cooled
with liquid nitrogen. Before the ground material thawed, extraction
buffer was added (50 mM sodium citrate, pH 6.5, 4 mM DTT, 2 mM calcium
chloride). Approx. 200 μl extraction buffer were added to approx. 100
mg (fresh weight) of plant material. Solid components of the suspension
of ground plant material and extraction buffer were separated by means of
centrifugation (10,000×g). An aliquot of the clear supernatant
obtained therefrom was mixed with a quarter of the extraction volume of
running buffer (40% glycerol, 250 mM Tris, pH 8.8, 0.02% bromophenol
blue) and separated in polyacrylamide gel (see below) at a constant
intensity of current of 20 mA per gel. (Before the protein extracts were
applied, an electrophoresis of the gels was carried out for 20 min under
the conditions indicated above). After the dye bromophenol blue in the
running buffer had run out of the gel, the electrophoresis was stopped.
Then, the gel was equilibrated five times in washing buffer (100 mM
sodium citrate, pH 6.5) at room temperature at a volume that was five
times the gel volume for 20 minutes each while stirring. Subsequently,
the gel was incubated in incubation buffer (100 mM sodium citrate, pH
6.5, 5% sucrose, 0.625 units of purified amylosucrase from Neisseria
polysaccharea (for purification of the enzyme and determination of the
activity see above)) in an amount that is five times the amount of the
gel volume at 30° C. for 16 hours. After decanting the incubation
buffer and after adding Lugol's solution (diluted at a ratio of 1:5), the
glucan which is formed by the amylosucrase in combination with the
branching enzyme becomes visible as bluish-brown band (FIG. 10). The
entire remaining polyacrylamide gel turns blue due to the amylosucrase
activity in the incubation buffer.

Composition of the Polyacrylamide Gel:

[0333] a) separation Gel

[0334] 375 mM Tris, pH 8.8

[0335] 7.5% polyacrylamide (Biorad no. EC-890)

[0336] for the polymerization:

[0337] 1/2000 volumes TEMED

[0338] 1/100 volumes ammonium persulfate

b) collection Gel

[0339] 125 mM Tris, pH 6.8

[0340] 4% polyacrylamide (Biorad no. EC-890)

[0341] for the polymerization:

[0342] 1/2000 volumes TEMED

[0343] 1/100 volumes ammonium persulfate

c) electrophoresis Buffer

[0344] 375 mM Tris, pH 8.8

[0345] 200 mM glycine

EXAMPLE 14

Analysis of the Starch of Plants Having an Increased Branching Enzyme
Activity

[0346] According to standard techniques, starch was isolated from
transgenic potato plants which had been produced according to Examples 12
and 13 and examined with regard to its physical and chemical properties.
It was found that the starch formed by the transgenic potato plants
differs from starch synthesized in wild type plants, for example in its
phosphate content and in the viscosity and pastification properties
determined by means of RVA. The results of the physico-chemical
characterisation of the modified starches based on the above-described
analysis techniques are shown in the following table.

[0347] The results of the RVA analysis, the analysis of the distribution
of the size of the starch granules and the gel texture are also shown in
FIGS. 11 to 15.

[0348] Furthermore, FIGS. 16 to 18 show the results of the HPLC
chromatographies which illustrate the pattern of the distribution of the
side-chains of the lines 143-WT (=wild type), 143-13A and 143-59A. FIG.
19 shows the elution gradient used in connection with the HPLC analysis.
In FIG. 20, the percentage deviation of side-chains having a certain
chain length from the wild type is shown.

[0349] The following two tables explain how the proportions of side-chains
were calculated.